HINT2

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005737 cytoplasm	IBA GO_REF:0000033	REMOVE	Summary: IBA annotation for cytoplasm based on phylogenetic inference. While the IBA includes cytoplasmic HINT family members (e.g., HINT1 is cytoplasmic), human HINT2 is specifically characterized as a mitochondrial protein. PMID:16762638 demonstrates clear mitochondrial localization via immunofluorescence, fractionation, and GFP-fusion. The cytoplasmic annotation may reflect family-level inference that does not apply to HINT2 specifically. Reason: Human HINT2 has an N-terminal mitochondrial targeting sequence and experimental evidence demonstrates exclusive mitochondrial localization. Unlike the cytoplasmic HINT1, HINT2 functions within mitochondria. The IBA inference from the broader HINT family does not apply to this paralog. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005739 mitochondrion	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for mitochondrion supported by phylogenetic inference including mouse Hint2 ortholog. This is strongly supported by experimental evidence from PMID:16762638 demonstrating mitochondrial localization by immunofluorescence co-localization with MitoTracker, mitochondrial fractionation, and GFP-fusion constructs. Reason: IBA annotation is well-supported by experimental literature. HINT2 has a validated N-terminal mitochondrial targeting sequence (~35 aa) and multiple lines of experimental evidence confirm mitochondrial localization. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005739 mitochondrion	IEA GO_REF:0000120	ACCEPT	Summary: IEA annotation based on automated transfer from mouse ortholog (UniProtKB:Q9D0S9). This is correct and consistent with experimental evidence from PMID:16762638. Reason: Automated annotation is correct. The orthologous mouse Hint2 and human HINT2 both localize to mitochondria, as established by direct experimental evidence. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005739 mitochondrion	IDA GO_REF:0000052	ACCEPT	Summary: IDA annotation from HPA (Human Protein Atlas) based on immunofluorescence data. This is consistent with the primary literature (PMID:16762638) showing mitochondrial localization. Reason: HPA immunofluorescence data confirms mitochondrial localization, consistent with the original characterization study. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005739 mitochondrion	HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an...	ACCEPT	Summary: HTP annotation from high-throughput mitochondrial proteome study (Morgenstern et al., 2021). This large-scale quantitative proteomics study provides high-confidence identification of HINT2 in the human mitochondrial proteome. Reason: High-throughput proteomics confirms mitochondrial localization, consistent with low-throughput experimental studies. Supporting Evidence: PMID:34800366 Quantitative high-confidence human mitochondrial proteome
GO:0005739 mitochondrion	IDA PMID:16762638 Hint2, a mitochondrial apoptotic sensitizer down-regulated i...	ACCEPT	Summary: Primary experimental evidence from Martin et al. (2006) demonstrating HINT2 mitochondrial localization via immunofluorescence with MitoTracker co-localization, mitochondrial fractionation, and GFP-fusion constructs. This is the foundational characterization of HINT2 subcellular localization. Reason: Gold standard experimental evidence establishing HINT2 as a mitochondrial protein. Multiple independent methods confirm localization. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005739 mitochondrion	ISS GO_REF:0000024	ACCEPT	Summary: ISS annotation based on sequence similarity to mouse Hint2 (UniProtKB:Q9D0S9). This is valid as both orthologs localize to mitochondria. Reason: Sequence similarity-based transfer is appropriate given the conserved N-terminal mitochondrial targeting sequence and experimental validation of localization. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria.
GO:0005759 mitochondrial matrix	IEA GO_REF:0000107	ACCEPT	Summary: IEA annotation for mitochondrial matrix based on Ensembl Compara transfer from mouse ortholog. This is consistent with the N-terminal mitochondrial targeting sequence (aa 1-17 per UniProt) which would direct the mature protein to the matrix after import and cleavage. The Reactome annotation suggesting outer membrane localization appears to be erroneous (see review of GO:0005741). Reason: The mitochondrial matrix localization is well-supported by the protein's N-terminal mitochondrial targeting sequence. UniProt annotates residues 1-17 as a transit peptide for mitochondrial import. After cleavage, the mature protein (aa 18-163) would be expected to reside in the matrix. This is consistent with HINT2's role in regulating mitochondrial NAD+ homeostasis and SIRT3-dependent deacetylation, as SIRT3 is a matrix-localized sirtuin. The Reactome outer membrane annotation appears to be erroneous and should not be used to question the matrix localization. The PMID:18653718 study also noted that HINT2 appeared "associated with mitochondrial membranes, probably facing the interior of the organelle" - consistent with matrix or inner membrane localization. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria. PMID:18653718 The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle.
GO:0005741 mitochondrial outer membrane	TAS Reactome:R-HSA-9693214	REMOVE	Summary: TAS annotation based on Reactome pathway R-HSA-9693214 claiming RHOD binds HINT2 at the mitochondrial outer membrane to trigger Ca2+ influx. The Reactome entry cites "Chen et al. 2017" and "Bagci et al. 2020". However, this annotation conflicts with multiple lines of evidence showing HINT2 has an N-terminal mitochondrial targeting sequence (aa 1-17 per UniProt) that directs the mature protein to the matrix. The Reactome interpretation may be erroneous or conflating HINT2 with a different protein. Reason: This annotation should be removed for several reasons. (1) HINT2 has a well-characterized N-terminal mitochondrial targeting sequence (aa 1-17 per UniProt FT TRANSIT) that directs the protein to the mitochondrial matrix, not the outer membrane. (2) The primary characterization study (PMID:16762638) localized HINT2 to mitochondria but did not demonstrate outer membrane localization specifically. (3) The Reactome citation to "Chen et al. 2017" for RHOD-HINT2 interaction triggering Ca2+ influx is questionable - more recent literature (e.g., PMC8677646) shows HINT2 interacts with MCU (mitochondrial calcium uniporter) which is in the inner membrane/matrix, not the outer membrane. The outer membrane localization may be a curation error conflating the Ca2+ signaling function with outer membrane localization. HINT2's N-terminal targeting sequence is incompatible with outer membrane localization of the mature protein. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria. Reactome:R-HSA-9693214 GTP-bound RHOD also binds HINT2 at the mitochondrial outer membrane, which triggers mitochondrial Ca2+ influx
GO:0016787 hydrolase activity	IBA GO_REF:0000033	MODIFY	Summary: IBA annotation for hydrolase activity based on phylogenetic inference across the HIT family. This is correct but overly general. HINT2 has specifically characterized adenosine 5'-monophosphoramidase activity (GO:0043530) which is a more informative term. Reason: While hydrolase activity is not incorrect, the more specific term GO:0043530 (adenosine 5'-monophosphoramidase activity) is experimentally established with detailed kinetics (kcat = 0.0223 s-1, Km = 128 uM) and should be preferred. Proposed replacements: adenosine 5'-monophosphoramidase activity Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L) PMID:31990367 Here, a similar substrate specificity profile (kcat /Km ) for model phosphoramidate substrates was found for hHINT2 but with higher kcat and Km values when compared with hHINT1
GO:0000166 nucleotide binding	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: IEA annotation for nucleotide binding based on UniProt keyword mapping. This is true but uninformative for a nucleotide-processing enzyme. The nucleotide (AMP) is a product of the enzymatic reaction, not a regulatory binding site. Reason: While HINT2 does bind nucleotides as part of its catalytic mechanism, this is inherent to its enzymatic function. The term "nucleotide binding" alone is uninformative and the more specific catalytic function (GO:0043530) better captures the biological role. Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group
GO:0003824 catalytic activity	IEA GO_REF:0000002	MODIFY	Summary: IEA annotation for generic catalytic activity based on InterPro domain mapping (HIT-like domain IPR011146). This is correct but extremely vague. The specific enzymatic activity (adenosine 5'-monophosphoramidase activity) is well-characterized. Reason: Generic "catalytic activity" should be replaced with the specific enzymatic function GO:0043530 (adenosine 5'-monophosphoramidase activity) which is experimentally validated. Proposed replacements: adenosine 5'-monophosphoramidase activity Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
GO:0016787 hydrolase activity	IEA GO_REF:0000043	MODIFY	Summary: IEA annotation for hydrolase activity based on UniProt keyword mapping. This is a duplicate of the concept captured by the IBA annotation. The term is correct but too general. Reason: Should use the more specific GO:0043530 (adenosine 5'-monophosphoramidase activity) which is experimentally established. Proposed replacements: adenosine 5'-monophosphoramidase activity Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
GO:0043530 adenosine 5'-monophosphoramidase activity	IEA GO_REF:0000120	ACCEPT	Summary: IEA annotation for the specific adenosine 5'-monophosphoramidase activity based on automated annotation (ARBA rule and Rhea reaction). This correctly captures the primary enzymatic function of HINT2. Reason: Correct specific annotation for HINT2's primary molecular function. This is the core enzymatic activity with well-characterized kinetics (kcat = 0.0223 s-1, Km = 128 uM for AMP-pNA substrate). Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
GO:0005515 protein binding	IPI PMID:35156780 CFTR interactome mapping using the mammalian membrane two-hy...	REMOVE	Summary: IPI annotation for protein binding with CFTR based on high-throughput mammalian membrane two-hybrid (MaMTH-HTS) screening. HINT2 is a mitochondrial matrix/inner membrane protein, while CFTR is a plasma membrane chloride channel. The physiological relevance of this interaction is highly questionable given the distinct subcellular compartments. Reason: This annotation should be removed because (1) "protein binding" is an uninformative term that does not specify the nature or consequence of the interaction, (2) the interaction was detected in a high-throughput artificial system (membrane two-hybrid) that may not reflect physiological conditions, and critically (3) HINT2 and CFTR localize to completely different subcellular compartments (mitochondria vs. plasma membrane), making physiological interaction extremely unlikely. HINT2 has an N-terminal mitochondrial targeting sequence and multiple studies confirm its mitochondrial localization (PMID:16762638). CFTR is a plasma membrane chloride channel. There is no plausible biological context where these proteins would interact in vivo. High-throughput protein-protein interaction screens frequently produce false positives, and this appears to be one. Supporting Evidence: PMID:16762638 Hint2 was localized in mitochondria. PMID:35156780 high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions
GO:0043530 adenosine 5'-monophosphoramidase activity	IDA PMID:16762638 Hint2, a mitochondrial apoptotic sensitizer down-regulated i...	ACCEPT	Summary: Primary experimental evidence from Martin et al. (2006) establishing HINT2 as an adenosine 5'-monophosphoramidase. Using the model substrate AMP-pNA, they determined kcat = 0.0223 s-1 and Km = 128 uM. The active site histidine (H149) was confirmed by mutagenesis - H149A mutation abolished enzymatic activity. Reason: Gold standard experimental evidence defining HINT2's core enzymatic function. This is the primary molecular function of HINT2 with detailed kinetic characterization and active site validation. Supporting Evidence: PMID:16762638 Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
GO:0043530 adenosine 5'-monophosphoramidase activity	IDA PMID:31990367 Histidine triad nucleotide-binding proteins HINT1 and HINT2 ...	ACCEPT	Summary: Experimental evidence from Strom et al. (2020) comparing substrate specificities between HINT1 and HINT2. They confirmed similar phosphoramidate hydrolysis activity for both enzymes, with HINT2 showing higher kcat and Km values compared to HINT1. Also demonstrated a broader pH range for maximum catalytic activity (pK1 = 6.76, pK2 = 8.41). Reason: Confirmatory experimental evidence for HINT2's adenosine 5'-monophosphoramidase activity with additional characterization of pH dependence and substrate specificity. Supporting Evidence: PMID:31990367 Here, a similar substrate specificity profile (kcat /Km ) for model phosphoramidate substrates was found for hHINT2 but with higher kcat and Km values when compared with hHINT1
GO:0006629 lipid metabolic process	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: IEA annotation for lipid metabolic process based on UniProt keyword mapping (KW-0443 Lipid metabolism). This is derived from correlative evidence in PMID:18653718 where HINT2 knockdown affected steroidogenic response in H295R adrenocortical cells. The connection to lipid metabolism is indirect and mechanistically unclear - the authors explicitly state the mechanism "still remains to determine at the molecular level." Reason: The evidence linking HINT2 to lipid metabolism is phenotypic and correlative, not mechanistic. PMID:18653718 shows that HINT2 knockdown reduced steroidogenic response, but the authors acknowledge the mechanism is unknown. More recent work (Wang et al. 2025) shows HINT2 deficiency exacerbates hepatic steatosis through effects on mitochondrial NAD+/SIRT3 pathways, but this represents an indirect consequence of disrupted mitochondrial function rather than direct involvement in lipid metabolic enzymes or pathways. HINT2's primary function is adenosine 5'-monophosphoramidase activity - there is no evidence it directly participates in lipid metabolic reactions. The term "lipid metabolic process" is too broad and implies direct involvement that is not supported. Supporting Evidence: PMID:18653718 these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level. file:human/HINT2/HINT2-deep-research-falcon.md HINT2 loss reduces mitochondrial NAD+ and SIRT3 activity; loss of Hint2 decreased mitochondrial NAD+ and SIRT3 activity, increased mitochondrial protein hyperacetylation, and exacerbated diet-induced steatosis
GO:0006694 steroid biosynthetic process	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: IEA annotation for steroid biosynthesis based on UniProt keyword mapping (KW-0752). This is derived from PMID:18653718 which showed that HINT2 knockdown in adrenocortical H295R cells reduced pregnenolone secretion in response to angiotensin II, K+, forskolin, or hydroxycholesterol stimulation. However, the mechanism is explicitly stated as unknown, and the effect appears to be through mitochondrial calcium signaling and membrane potential rather than direct participation in steroidogenic enzymes. Reason: The evidence from Lenglet et al. (2008) is correlative and the mechanism is explicitly unknown. The abstract states that HINT2's effects "could be related to its ability to maintain a favorable mitochondrial potential" and that the mechanism "still remains to determine at the molecular level." Importantly, HINT2 overexpression had "no effect" on steroidogenesis - only knockdown showed effects, suggesting HINT2 is not rate-limiting but rather maintains basal mitochondrial function required for optimal steroidogenic capacity. HINT2 is NOT a steroidogenic enzyme (like CYP11A1, HSD3B, etc.) - it is an adenosine 5'-monophosphoramidase. The steroidogenic effects are likely secondary to disruption of mitochondrial calcium handling or membrane potential rather than direct involvement in steroid biosynthetic pathways. This represents classic over-annotation from phenotypic observations. Supporting Evidence: PMID:18653718 these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level. PMID:18653718 Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. PMID:18653718 Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential.
GO:0006915 apoptotic process	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	Summary: IEA annotation for apoptotic process based on UniProt keyword mapping (KW-0053). The original evidence from PMID:16762638 shows HINT2 overexpression sensitizes hepatoma cells to Fas-mediated apoptosis (reduced viability, enhanced mitochondrial depolarization, increased caspase cleavage). However, this is a PHENOTYPIC observation in a specific cell model, not evidence of direct mechanistic involvement in the apoptotic machinery. The authors describe HINT2 as an "apoptotic sensitizer" - meaning it modulates sensitivity to apoptotic stimuli, likely through effects on mitochondrial function (membrane potential, NAD+ homeostasis), rather than being a core component of apoptotic signaling. Reason: The evidence from Martin et al. (2006) demonstrates that HINT2 modulates cellular sensitivity to apoptotic stress, but does not establish HINT2 as a direct participant in apoptotic signaling pathways. The observed effects (mitochondrial depolarization, caspase activation) are downstream consequences of HINT2's role in mitochondrial function, not evidence of direct apoptotic function. The abstract explicitly states the "biological function" of HINT2 is "uncertain" and classifies it as an "apoptotic sensitizer" rather than an apoptotic regulator. More recent studies (Wang et al. 2025, Fan et al. 2020) link HINT2 to mitochondrial NAD+ homeostasis and SIRT3-dependent deacetylation, suggesting the apoptotic phenotype is secondary to bioenergetic roles. GO annotation for "apoptotic process" implies direct involvement, which is not supported by the mechanistic evidence. Supporting Evidence: PMID:16762638 Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. PMID:16762638 Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma. file:human/HINT2/HINT2-deep-research-falcon.md HINT2 modulates mitochondrial NAD+ homeostasis and promotes SIRT3-dependent deacetylation; overexpression increases mitochondrial NAD+ and reduces mitochondrial protein hyperacetylation

Core Functions

Adenosine 5'-monophosphoramidase activity - hydrolyzes purine nucleotide phosphoramidates with a single phosphate group (e.g., AMP-NH2 to AMP + NH2). Catalytic activity depends on the conserved His-X-His-X-His histidine triad motif, with His-149 essential for catalysis (kcat = 0.0223 s-1, Km = 128 uM for AMP-pNA substrate).

Molecular Function:

adenosine 5'-monophosphoramidase activity

Cellular Locations:

mitochondrion

Supporting Evidence:

PMID:16762638
Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
PMID:31990367
Here, a similar substrate specificity profile (kcat /Km ) for model phosphoramidate substrates was found for hHINT2

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000024

Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000043

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

GO_REF:0000052

Gene Ontology annotation based on curation of immunofluorescence data

GO_REF:0000107

Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:16762638

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma.

HINT2 is localized to mitochondria

"Hint2 was localized in mitochondria."
Exhibits adenosine 5'-monophosphoramidase activity

"Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)"
Catalysis depends on conserved histidine triad

"Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases"
Functions as a mitochondrial apoptotic sensitizer

"Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable"
Highly expressed in liver and pancreas

"Hint2 was predominantly expressed in liver and pancreas"
Downregulated in hepatocellular carcinoma

"HINT2 messenger RNA is downregulated in hepatocellular carcinomas"

PMID:18653718

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis.

HINT2 is expressed in mitochondria of adrenocortical H295R cells

"Hint2 is expressed in the mitochondria of H295R cells"
HINT2 is involved in steroidogenesis

"this protein is involved in steroidogenesis"

PMID:31990367

Histidine triad nucleotide-binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A.

HINT1 and HINT2 have similar substrate specificity profiles

"Here, a similar substrate specificity profile (kcat /Km ) for model phosphoramidate substrates was found for hHINT2 but with higher kcat and Km values when compared with hHINT1"
HINT2 has higher kcat and Km values compared to HINT1

"with higher kcat and Km values when compared with hHINT1"
HINT2 has a broader pH range for maximum catalytic activity

"A broader pH range for maximum catalytic activity was determined for hHINT2 (pK1 = 6.76 ± 0.16, pK2 = 8.41 ± 0.07)"
Neither HINT1 nor HINT2 bind Ap4A

"Ap4 A was found to have no detectable binding to HINT1 nor HINT2 by isothermal titration calorimetry"

PMID:34800366

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.

HINT2 identified in high-confidence human mitochondrial proteome by quantitative proteomics

"mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP)"

PMID:35156780

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

HINT2 detected as CFTR interactor in high-throughput screening (physiological relevance unclear)

"high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions"

Reactome:R-HSA-9693214

RHOD binds effectors at the mitochondrial outer membrane

GTP-bound RHOD binds HINT2 at mitochondrial outer membrane

"GTP-bound RHOD also binds HINT2 at the mitochondrial outer membrane"
This interaction triggers mitochondrial Ca2+ influx

"which triggers mitochondrial Ca2+ influx"

file:human/HINT2/HINT2-deep-research-falcon.md

Deep research summary for HINT2 functional annotation

HINT2 regulates mitochondrial NAD+ homeostasis and SIRT3-dependent deacetylation
HINT2 overexpression is cardioprotective after myocardial infarction
HINT2 is downregulated in hepatocellular carcinoma and colorectal cancer

Suggested Experiments

Experiment: Determine sub-mitochondrial localization using protease protection assays to clarify whether HINT2 is in the matrix (as predicted by targeting sequence) or accessible at the outer membrane (as suggested by RHOD interaction data).

Experiment: Identify endogenous substrates using metabolomics by comparing metabolite profiles in HINT2 knockout vs wildtype mitochondria to identify accumulating phosphoramidates that may be endogenous substrates.

Deep Research

Falcon

(HINT2-deep-research-falcon.md)

this with annotations you find in gene/protein databases, but these can be outdated or inaccurate. Falcon Edison Scientific Literature 18 citations 2025-12-20T14:40:24.066144

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Comprehensive Research Report: Human HINT2 (UniProt Q9BX68)

Summary and identity verification
- Gene/protein identity and domains: HINT2 encodes a histidine triad (HIT) family hydrolase with a conserved His–X–His–X–His catalytic motif and an N‑terminal mitochondrial targeting sequence. Human HINT2 localizes to mitochondria and is a 163‑amino‑acid protein, consistent with the UniProt entry for Q9BX68 (human). Mitochondrial localization was demonstrated by immunofluorescence co‑localization with MitoTracker, mitochondrial fractionation, and GFP‑fusion constructs; mutation of the middle histidine (H149A) abolished enzymatic function, confirming the HIT active site’s role (Martin et al., 2006; Gastroenterology; published June 2006; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 1-2, martin2006hint2amitochondrial pages 6-7).

Key concepts and definitions
- Primary biochemical function: HINT2 is an adenosine 5′‑monophosphoramidase (EC 3.9.1.-) consistent with HIT family hydrolases. Using the model substrate AMP‑pNA, human HINT2 exhibits kcat = 0.0223 ± 0.0031 s−1 and Km = 128 ± 35 µM; activity depends on the conserved histidine triad (H149A mutant abolishes activity). These data establish nucleoside monophosphoramidase activity and implicate the histidine triad in catalysis (Martin et al., 2006; Gastroenterology; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 1-2, martin2006hint2amitochondrial pages 6-7).
- Subcellular localization and tissue expression: HINT2 is localized to mitochondria and is most abundantly expressed in liver and pancreas (Martin et al., 2006; Gastroenterology; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 1-2).
- Functional role in apoptosis: In hepatoma cells, HINT2 acts as a mitochondrial apoptotic sensitizer. Overexpression in HepG2 cells increased sensitivity to anti‑Fas + actinomycin D, reducing viable cells and enhancing mitochondrial depolarization and caspase cleavage; xenografts overexpressing HINT2 formed smaller tumors with increased apoptosis (Martin et al., 2006; Gastroenterology; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7).

Recent developments and latest research (priority to 2023–2024 where possible)
- Mitochondrial NAD+ homeostasis and SIRT3 axis: New mechanistic work demonstrates HINT2 enhances mitochondrial NAD+ availability and SIRT3‑dependent deacetylation in liver disease models. In mouse and cell systems, loss of Hint2 decreased mitochondrial NAD+ and SIRT3 activity, increased mitochondrial protein hyperacetylation, and exacerbated diet‑induced steatosis; HINT2 overexpression increased mitochondrial NAD+, reduced triglycerides, and improved mitochondrial function. These effects were linked to the mitochondrial NAD+ carrier SLC25A51 (NDTM1), whose modulation shifted mitochondrial vs cytosolic NAD+ pools; HINT2 increased SLC25A51 protein indirectly (no co‑IP). AMPK activity changes were implicated in this regulation (Wang et al., 2025; Exp. & Mol. Med.; published May 2025; https://doi.org/10.1038/s12276-025-01445-w) (wang2025histidinetriadnucleotidebinding pages 9-11).
- Cardiomyocyte protection after MI and NMN dependence: In a post‑MI mouse model, AAV9‑mediated HINT2 overexpression preserved cardiac function, increased ATP production, improved metabolic readouts, and reduced apoptosis and fibrosis. HINT2 raised mitochondrial NAD+ in neonatal mouse ventricular myocytes under hypoxia; its protective effect required NAMPT activity or exogenous NMN. Biophysical assays supported direct HINT2–NMN binding (Fan et al., 2020; Acta Physiologica; published Jan 2020; https://doi.org/10.1111/apha.13439) (fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9).
- Note on 2023–2024 literature: Within the present evidence set, the most up‑to‑date mechanistic study is 2025 (Exp. & Mol. Med.) on the HINT2–NAD+–SIRT3–SLC25A51 axis. Contemporary 2023–2024 references could not be validated in the current context; therefore, conclusions emphasize directly supported studies (wang2025histidinetriadnucleotidebinding pages 9-11, fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9).

Current applications and real‑world implementations
- Prognostic biomarker potential in HCC: Transcriptomic analyses indicate HINT2 is down‑regulated in HCC, and higher HINT2 expression correlates with longer recurrence‑free survival. In a TCGA‑based analysis, high HINT2 was associated with reduced recurrence risk (univariate HR 0.567, 95% CI 0.339–0.950, P = .031; multivariate HR 0.560, 95% CI 0.317–0.991, P = .047). Clinical translation remains investigational due to limited sample sizes and lack of circulating biomarker detectability, but IHC assessment in tumor tissues is recommended (Zhou et al., 2019; Medicine; published Nov 2019; https://doi.org/10.1097/md.0000000000017815) (zhou2019clinicalsignificanceof pages 3-4).
- Therapeutic concepts in cardiology and liver disease: Preclinical AAV‑HINT2 delivery post‑MI and metabolic modulation via NAD+ (e.g., NMN) illustrate potential strategies to support mitochondrial bioenergetics; in hepatic models, restoring HINT2 function or downstream NAD+/SIRT3 pathways mitigated steatosis and improved mitochondrial function (Fan et al., 2020; https://doi.org/10.1111/apha.13439; Wang et al., 2025; https://doi.org/10.1038/s12276-025-01445-w) (fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9, wang2025histidinetriadnucleotidebinding pages 9-11).

Expert opinions and analysis from authoritative sources
- Mechanistic integration (apoptosis and bioenergetics): Early work situated HINT2 as a mitochondrial apoptotic sensitizer in liver cancer models, implicating mitochondrial depolarization and caspase activation without changes in cytochrome c release or Bcl‑2 phosphorylation under the tested conditions (Martin et al., 2006; Gastroenterology; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7). Later studies connect HINT2 to mitochondrial NAD+ handling and SIRT3‑mediated deacetylation, providing a unifying framework where HINT2 can influence both apoptotic sensitivity and mitochondrial protein acylation states through NAD+ availability (Fan et al., 2020; Wang et al., 2025) (fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9, wang2025histidinetriadnucleotidebinding pages 9-11).
- Pathway context and transport: The data support a functional link between HINT2 and mitochondrial NAD+ influx mediated by SLC25A51, although direct physical interaction was not detected (implying upstream regulatory control). AMPK emerges as a candidate intermediary modulated by HINT2 in hepatocytes (Wang et al., 2025; https://doi.org/10.1038/s12276-025-01445-w) (wang2025histidinetriadnucleotidebinding pages 9-11).

Relevant statistics and data from recent studies
- Enzymology and catalytic dependence:
• AMP‑pNA hydrolysis: kcat 0.0223 ± 0.0031 s−1; Km 128 ± 35 µM; abolished by H149A mutation (Martin et al., 2006; https://doi.org/10.1053/j.gastro.2006.03.024) (martin2006hint2amitochondrial pages 1-2, martin2006hint2amitochondrial pages 6-7).
- Apoptosis and tumor suppression in vitro/in vivo (hepatoma):
• Anti‑Fas + actinomycin D viability: 32.2% ± 0.6% (HINT2 OE) vs 57.7% ± 4.6% (control), P < .02; mitochondrial depolarization at 16 h: 87.8% ± 2.3% vs 49.7% ± 1.6%, P < .05; xenograft tumor weight: 0.32 ± 0.13 g vs 0.85 ± 0.35 g, P < .05 (Martin et al., 2006) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7).
- HCC prognosis:
• Recurrence‑free survival HRs: univariate 0.567 (95% CI 0.339–0.950), P = .031; multivariate 0.560 (95% CI 0.317–0.991), P = .047 (Zhou et al., 2019; https://doi.org/10.1097/md.0000000000017815) (zhou2019clinicalsignificanceof pages 3-4).
- Metabolic liver disease mechanisms (MASLD):
• HINT2 loss reduces mitochondrial NAD+ and SIRT3 activity; NMN (500 mg/kg i.p. every 2 days) restored mitochondrial NAD+/SIRT3 activity and improved hepatic lipids in Hint2‑deficient settings. SLC25A51 overexpression lowered cellular triglycerides; SLC25A51 knockdown shifted NAD+ away from mitochondria (Wang et al., 2025; https://doi.org/10.1038/s12276-025-01445-w) (wang2025histidinetriadnucleotidebinding pages 9-11).
- Cardioprotection post‑MI and NAD+ handling:
• HINT2 overexpression improved cardiac ATP, increased mitochondrial NAD+ in hypoxic cardiomyocytes, and reduced apoptosis/fibrosis. HINT2’s benefit required NAMPT or NMN; direct NMN–HINT2 binding supported by biolayer interferometry (Fan et al., 2020; https://doi.org/10.1111/apha.13439) (fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9).

Disease associations and mechanistic roles beyond HCC
- Colorectal cancer (CRC): HINT2 is downregulated in primary CRC and metastatic lesions, associated with stage progression. Functional experiments showed that HINT2 loss increased migration/invasion and promoted liver metastasis in vivo. Mechanistically, HINT2 downregulation induced EMT via HIF‑2α‑dependent transcriptional activation of ZEB1; ZEB1 silencing rescued CDH1 repression and migration/invasion phenotypes (Li et al., 2017; Oncotarget; published Jan 2017; https://doi.org/10.18632/oncotarget.14587). Note: journal quality caveat applies to Oncotarget publications; findings should be independently validated (li2017hint2downregulationpromotes pages 1-3, li2017hint2downregulationpromotes pages 5-7).
- Metabolic liver disease (steatosis, acetylation): Evidence links HINT2 deficiency to mitochondrial protein hyperacetylation and worsened steatotic phenotypes. Restoring mitochondrial NAD+ and SIRT3 activity ameliorates these outcomes (Wang et al., 2025; https://doi.org/10.1038/s12276-025-01445-w) (wang2025histidinetriadnucleotidebinding pages 9-11).
- Cardiac injury: Post‑MI AAV9‑HINT2 delivery improved metabolic and functional outcomes in mice, implicating mitochondrial NAD+ maintenance as a protective mechanism (Fan et al., 2020; https://doi.org/10.1111/apha.13439) (fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9).

Clarifications, limitations, and open questions
- MCU/calcium handling: Direct experimental evidence for physical interaction between HINT2 and the MCU complex is not present in the current evidence set. While HINT2’s mitochondrial role suggests potential crosstalk with calcium signaling, specific MCU/HINT2 mechanisms require further study and are not concluded here (no supporting context IDs).
- 2023–2024 update emphasis: Aside from the 2025 mechanistic paper, few 2023–2024 HINT2‑focused primary studies were recoverable in the present context. The core conclusions therefore rely on 2006–2020 primary data and the 2025 mechanistic advance; newer studies should be incorporated as they become available (wang2025histidinetriadnucleotidebinding pages 9-11, martin2006hint2amitochondrial pages 10-17, fan2020overexpressionofthe pages 11-14, martin2006hint2amitochondrial pages 6-7).

Embedded evidence summary table
| Aspect | Evidence summary | Key quantitative data | Source |
|---|---|---:|---|
| Identity & domains | Member of the HIT (histidine triad) family with an N-terminal mitochondrial targeting sequence (~35 aa) and a ~163-amino-acid polypeptide length. | Length ≈163 aa; N-terminal MTS ≈35 aa; conserved HIT motif present. | (martin2006hint2amitochondrial pages 10-17) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Enzymatic activity | Shows adenosine 5'-monophosphoramidase activity measured using AMP-pNA (model substrate), consistent with HIT-family hydrolase function. | kcat = 0.0223 ± 0.0031 s^-1; Km = 128 ± 35 μM (AMP-pNA) (reported vs rabbit Hint1 kcat = 0.00187 s^-1). | (martin2006hint2amitochondrial pages 1-2, martin2006hint2amitochondrial pages 6-7) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Catalytic motif / residue | Catalysis depends on the conserved His–X–His–X–His (HIT) motif; middle histidine (H149) is essential (mutation abolishes activity). | H149A mutant abrogates detectable AMP-pNA hydrolysis. | (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Subcellular localization | Robust mitochondrial localization demonstrated by immunofluorescence (colocalization with MitoTracker), mitochondrial fractionation, and GFP-fusion targeting experiments. | Mitochondrial matrix/mitochondrial localization confirmed by multiple assays. | (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 1-2) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Tissue expression | Predominant expression in liver and pancreas by qRT-PCR and immunoblot analyses. | High expression in liver (tissue panel data reported). | (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 1-2) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Functional role — apoptotic sensitization | Overexpression in HepG2 cells sensitizes cells to death-receptor (anti-Fas + actinomycin D) induced apoptosis; knockdown reduces apoptotic responses. | Viability after anti-Fas+actD: 32.2% ± 0.6% (HINT2 OE) vs 57.7% ± 4.6% (control); mitochondrial depolarization at 16 h: 87.8% ± 2.3% vs 49.7% ± 1.6%; xenograft tumor weight: 0.32 ± 0.13 g vs 0.85 ± 0.35 g. | (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7) DOI: https://doi.org/10.1053/j.gastro.2006.03.024 |
| Bioenergetics — NAD+, SIRT3, acetylation | HINT2 modulates mitochondrial NAD+ homeostasis and promotes SIRT3-dependent deacetylation; overexpression increases mitochondrial NAD+ and reduces mitochondrial protein hyperacetylation, while loss causes hyperacetylation and worsens metabolic phenotypes. | NMN rescue in mice: NMN 500 mg/kg i.p. every 2 days (restored mitochondrial NAD+ and SIRT3 activity in models); computational/biophysical HINT2–NMN binding support (docking/biolayer interferometry R2 ≈ 0.9842). | (wang2025histidinetriadnucleotidebinding pages 9-11, fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9) DOI: https://doi.org/10.1038/s12276-025-01445-w, https://doi.org/10.1111/apha.13439 |
| Link to mitochondrial NAD import (SLC25A51) | HINT2 increases mitochondrial SLC25A51 protein (indirectly) and SLC25A51 manipulations shift mitochondrial vs cytosolic NAD+ pools; HINT2 does not co-immunoprecipitate with SLC25A51, suggesting indirect regulation (possible AMPK involvement). | SLC25A51 overexpression reduced intracellular TG; knockdown ↑cytosolic NAD+ and ↓mitochondrial NAD+; no stable HINT2–SLC25A51 co-IP reported. | (wang2025histidinetriadnucleotidebinding pages 9-11) DOI: https://doi.org/10.1038/s12276-025-01445-w |
| Disease relevance (HCC, MASLD/MASH, post-MI heart) | HINT2 is downregulated in HCC and associated with poorer prognosis; HINT2 loss exacerbates diet-induced steatosis (MASLD models) and HINT2 overexpression is cardioprotective after MI in mice. | HCC microarray: HINT2 downregulation −0.424 ± 0.579 vs −0.109 ± 0.277 (log2); 77% of HCCs lower than normal; poor‑prognosis subclass −0.728 ± 0.522 vs −0.187 ± 0.506 (P < 1e-5). Recurrence-free survival: univariate HR 0.567 (95% CI 0.339–0.950, P = 0.031); multivariate HR 0.560 (95% CI 0.317–0.991, P = 0.047). MASLD/MI: Hint2 KO worsens steatosis; AAV9‑HINT2 improves ATP/NAD and reduces apoptosis/fibrosis after MI. | (martin2006hint2amitochondrial pages 6-7, zhou2019clinicalsignificanceof pages 3-4, wang2025histidinetriadnucleotidebinding pages 9-11, fan2020overexpressionofthe pages 11-14) DOIs: https://doi.org/10.1053/j.gastro.2006.03.024; https://doi.org/10.1097/md.0000000000017815; https://doi.org/10.1038/s12276-025-01445-w; https://doi.org/10.1111/apha.13439 |

Table: Concise, evidence‑mapped summary of human HINT2 (UniProt Q9BX68) covering identity, enzymology, localization, functions (apoptosis and mitochondrial NAD/SIRT3 regulation), links to SLC25A51, and disease associations with key quantitative data and sources (context IDs shown).

Conclusions
Human HINT2 (Q9BX68) is a mitochondrial HIT‑family adenosine 5′‑monophosphoramidase whose activity depends on the histidine triad, with strong expression in liver and pancreas. Functionally, HINT2 sensitizes hepatocytes to apoptosis and regulates mitochondrial bioenergetics by promoting mitochondrial NAD+ availability, SIRT3 activity, and balanced protein acetylation. In cancer, HINT2 is downregulated in HCC and CRC, with prognostic implications in HCC and EMT regulation in CRC via HIF‑2α/ZEB1. In cardiometabolic disease models, augmenting HINT2 improves post‑MI cardiac outcomes and mitigates hepatic steatosis through NAD+/SIRT3 pathways. Together, these findings prioritize HINT2 as a mitochondrial regulator with translational potential in oncology and metabolic/cardiac disease, with current therapeutic hypotheses centered on restoring HINT2 expression or leveraging its NAD+‑linked mechanisms (Martin et al., 2006; Zhou et al., 2019; Fan et al., 2020; Wang et al., 2025) (martin2006hint2amitochondrial pages 10-17, martin2006hint2amitochondrial pages 6-7, zhou2019clinicalsignificanceof pages 3-4, fan2020overexpressionofthe pages 11-14, fan2020overexpressionofthe pages 6-9, wang2025histidinetriadnucleotidebinding pages 9-11).

References

(martin2006hint2amitochondrial pages 10-17): Juliette Martin, Fabrice Magnino, Karin Schmidt, Anne–Christine Piguet, Ju–Seog Lee, David Semela, Marie V. St–Pierre, Andrew Ziemiecki, Doris Cassio, Charles Brenner, Snorri S. Thorgeirsson, and Jean–François Dufour. Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma. Gastroenterology, 130 7:2179-88, Jun 2006. URL: https://doi.org/10.1053/j.gastro.2006.03.024, doi:10.1053/j.gastro.2006.03.024. This article has 77 citations and is from a highest quality peer-reviewed journal.
(martin2006hint2amitochondrial pages 1-2): Juliette Martin, Fabrice Magnino, Karin Schmidt, Anne–Christine Piguet, Ju–Seog Lee, David Semela, Marie V. St–Pierre, Andrew Ziemiecki, Doris Cassio, Charles Brenner, Snorri S. Thorgeirsson, and Jean–François Dufour. Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma. Gastroenterology, 130 7:2179-88, Jun 2006. URL: https://doi.org/10.1053/j.gastro.2006.03.024, doi:10.1053/j.gastro.2006.03.024. This article has 77 citations and is from a highest quality peer-reviewed journal.
(martin2006hint2amitochondrial pages 6-7): Juliette Martin, Fabrice Magnino, Karin Schmidt, Anne–Christine Piguet, Ju–Seog Lee, David Semela, Marie V. St–Pierre, Andrew Ziemiecki, Doris Cassio, Charles Brenner, Snorri S. Thorgeirsson, and Jean–François Dufour. Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma. Gastroenterology, 130 7:2179-88, Jun 2006. URL: https://doi.org/10.1053/j.gastro.2006.03.024, doi:10.1053/j.gastro.2006.03.024. This article has 77 citations and is from a highest quality peer-reviewed journal.
(wang2025histidinetriadnucleotidebinding pages 9-11): Qinqiu Wang, Yanjun Guo, Shenghui Chen, Zhening Liu, Xinyu Wang, Hangkai Huang, Qi-en Shen, Ling Yang, Meng Li, Youming Li, Chaohui Yu, and Chengfu Xu. Histidine triad nucleotide-binding protein 2 attenuates metabolic dysfunction-associated steatotic liver disease through nad+-dependent sirtuin-3 activation. Experimental & Molecular Medicine, 57:990-1004, May 2025. URL: https://doi.org/10.1038/s12276-025-01445-w, doi:10.1038/s12276-025-01445-w. This article has 0 citations and is from a peer-reviewed journal.
(fan2020overexpressionofthe pages 11-14): Mengkang Fan, Zhangwei Chen, Yin Huang, Yan Xia, Ao Chen, Danbo Lu, Yuan Wu, Ning Zhang, Peipei Zhang, Su Li, Jinxiang Chen, Yingmei Zhang, Aijun Sun, Yunzeng Zou, Kai Hu, Juying Qian, and Junbo Ge. Overexpression of the histidine triad nucleotide‐binding protein 2 protects cardiac function in the adult mice after acute myocardial infarction. Acta Physiologica, Jan 2020. URL: https://doi.org/10.1111/apha.13439, doi:10.1111/apha.13439. This article has 20 citations and is from a domain leading peer-reviewed journal.
(fan2020overexpressionofthe pages 6-9): Mengkang Fan, Zhangwei Chen, Yin Huang, Yan Xia, Ao Chen, Danbo Lu, Yuan Wu, Ning Zhang, Peipei Zhang, Su Li, Jinxiang Chen, Yingmei Zhang, Aijun Sun, Yunzeng Zou, Kai Hu, Juying Qian, and Junbo Ge. Overexpression of the histidine triad nucleotide‐binding protein 2 protects cardiac function in the adult mice after acute myocardial infarction. Acta Physiologica, Jan 2020. URL: https://doi.org/10.1111/apha.13439, doi:10.1111/apha.13439. This article has 20 citations and is from a domain leading peer-reviewed journal.
(zhou2019clinicalsignificanceof pages 3-4): Dong-Kai Zhou, Xiao-Hui Qian, Jun Cheng, Ling-Hui Chen, and Wei-Lin Wang. Clinical significance of down-regulated hint2 in hepatocellular carcinoma. Medicine, 98:e17815, Nov 2019. URL: https://doi.org/10.1097/md.0000000000017815, doi:10.1097/md.0000000000017815. This article has 11 citations and is from a peer-reviewed journal.
(li2017hint2downregulationpromotes pages 1-3): Weihua Li, Shaoxin Cai, Le Wang, Changshun Yang, Biaohuan Zhou, and Huan Wang. Hint2 downregulation promotes colorectal carcinoma migration and metastasis. Oncotarget, 8:13521-13531, Jan 2017. URL: https://doi.org/10.18632/oncotarget.14587, doi:10.18632/oncotarget.14587. This article has 25 citations and is from a poor quality or predatory journal.
(li2017hint2downregulationpromotes pages 5-7): Weihua Li, Shaoxin Cai, Le Wang, Changshun Yang, Biaohuan Zhou, and Huan Wang. Hint2 downregulation promotes colorectal carcinoma migration and metastasis. Oncotarget, 8:13521-13531, Jan 2017. URL: https://doi.org/10.18632/oncotarget.14587, doi:10.18632/oncotarget.14587. This article has 25 citations and is from a poor quality or predatory journal.

Citations

wang2025histidinetriadnucleotidebinding pages 9-11
zhou2019clinicalsignificanceof pages 3-4
fan2020overexpressionofthe pages 11-14
fan2020overexpressionofthe pages 6-9
https://doi.org/10.1053/j.gastro.2006.03.024
https://doi.org/10.1038/s12276-025-01445-w
https://doi.org/10.1111/apha.13439
https://doi.org/10.1097/md.0000000000017815
https://doi.org/10.1111/apha.13439;
https://doi.org/10.18632/oncotarget.14587
https://doi.org/10.1038/s12276-025-01445-w,
https://doi.org/10.1053/j.gastro.2006.03.024;
https://doi.org/10.1097/md.0000000000017815;
https://doi.org/10.1038/s12276-025-01445-w;
https://doi.org/10.1053/j.gastro.2006.03.024,
https://doi.org/10.1111/apha.13439,
https://doi.org/10.1097/md.0000000000017815,
https://doi.org/10.18632/oncotarget.14587,

📄 View Raw YAML

id: Q9BX68
gene_symbol: HINT2
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: HINT2 (Histidine triad nucleotide-binding protein 2) is a mitochondrial
  adenosine 5'-monophosphoramidase belonging to the HIT (histidine triad) superfamily.
  The enzyme hydrolyzes purine nucleotide phosphoramidates with a single phosphate
  group, using the conserved His-X-His-X-His catalytic motif. HINT2 localizes to mitochondria
  where it functions as an apoptotic sensitizer and regulator of mitochondrial NAD+
  homeostasis, influencing SIRT3-dependent protein deacetylation. The protein is highly
  expressed in liver and pancreas, and is downregulated in hepatocellular carcinoma
  (HCC) where low expression correlates with poor prognosis.
existing_annotations:
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for cytoplasm based on phylogenetic inference. While the
      IBA includes cytoplasmic HINT family members (e.g., HINT1 is cytoplasmic), human
      HINT2 is specifically characterized as a mitochondrial protein. PMID:16762638
      demonstrates clear mitochondrial localization via immunofluorescence, fractionation,
      and GFP-fusion. The cytoplasmic annotation may reflect family-level inference
      that does not apply to HINT2 specifically.
    action: REMOVE
    reason: Human HINT2 has an N-terminal mitochondrial targeting sequence and experimental
      evidence demonstrates exclusive mitochondrial localization. Unlike the cytoplasmic
      HINT1, HINT2 functions within mitochondria. The IBA inference from the broader
      HINT family does not apply to this paralog.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for mitochondrion supported by phylogenetic inference
      including mouse Hint2 ortholog. This is strongly supported by experimental evidence
      from PMID:16762638 demonstrating mitochondrial localization by immunofluorescence
      co-localization with MitoTracker, mitochondrial fractionation, and GFP-fusion
      constructs.
    action: ACCEPT
    reason: IBA annotation is well-supported by experimental literature. HINT2 has
      a validated N-terminal mitochondrial targeting sequence (~35 aa) and multiple
      lines of experimental evidence confirm mitochondrial localization.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: IEA annotation based on automated transfer from mouse ortholog (UniProtKB:Q9D0S9).
      This is correct and consistent with experimental evidence from PMID:16762638.
    action: ACCEPT
    reason: Automated annotation is correct. The orthologous mouse Hint2 and human
      HINT2 both localize to mitochondria, as established by direct experimental evidence.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  review:
    summary: IDA annotation from HPA (Human Protein Atlas) based on immunofluorescence
      data. This is consistent with the primary literature (PMID:16762638) showing
      mitochondrial localization.
    action: ACCEPT
    reason: HPA immunofluorescence data confirms mitochondrial localization, consistent
      with the original characterization study.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HTP
  original_reference_id: PMID:34800366
  review:
    summary: HTP annotation from high-throughput mitochondrial proteome study (Morgenstern
      et al., 2021). This large-scale quantitative proteomics study provides high-confidence
      identification of HINT2 in the human mitochondrial proteome.
    action: ACCEPT
    reason: High-throughput proteomics confirms mitochondrial localization, consistent
      with low-throughput experimental studies.
    supported_by:
    - reference_id: PMID:34800366
      supporting_text: Quantitative high-confidence human mitochondrial proteome
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: PMID:16762638
  review:
    summary: Primary experimental evidence from Martin et al. (2006) demonstrating
      HINT2 mitochondrial localization via immunofluorescence with MitoTracker co-localization,
      mitochondrial fractionation, and GFP-fusion constructs. This is the foundational
      characterization of HINT2 subcellular localization.
    action: ACCEPT
    reason: Gold standard experimental evidence establishing HINT2 as a mitochondrial
      protein. Multiple independent methods confirm localization.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: ISS annotation based on sequence similarity to mouse Hint2 (UniProtKB:Q9D0S9).
      This is valid as both orthologs localize to mitochondria.
    action: ACCEPT
    reason: Sequence similarity-based transfer is appropriate given the conserved
      N-terminal mitochondrial targeting sequence and experimental validation of localization.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
- term:
    id: GO:0005759
    label: mitochondrial matrix
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  review:
    summary: IEA annotation for mitochondrial matrix based on Ensembl Compara transfer
      from mouse ortholog. This is consistent with the N-terminal mitochondrial
      targeting sequence (aa 1-17 per UniProt) which would direct the mature protein
      to the matrix after import and cleavage. The Reactome annotation suggesting
      outer membrane localization appears to be erroneous (see review of GO:0005741).
    action: ACCEPT
    reason: The mitochondrial matrix localization is well-supported by the protein's
      N-terminal mitochondrial targeting sequence. UniProt annotates residues 1-17
      as a transit peptide for mitochondrial import. After cleavage, the mature
      protein (aa 18-163) would be expected to reside in the matrix. This is
      consistent with HINT2's role in regulating mitochondrial NAD+ homeostasis
      and SIRT3-dependent deacetylation, as SIRT3 is a matrix-localized sirtuin.
      The Reactome outer membrane annotation appears to be erroneous and should
      not be used to question the matrix localization. The PMID:18653718 study
      also noted that HINT2 appeared "associated with mitochondrial membranes,
      probably facing the interior of the organelle" - consistent with matrix or
      inner membrane localization.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
    - reference_id: PMID:18653718
      supporting_text: The protein appeared associated with mitochondrial membranes,
        probably facing the interior of the organelle.
- term:
    id: GO:0005741
    label: mitochondrial outer membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9693214
  review:
    summary: TAS annotation based on Reactome pathway R-HSA-9693214 claiming RHOD
      binds HINT2 at the mitochondrial outer membrane to trigger Ca2+ influx. The
      Reactome entry cites "Chen et al. 2017" and "Bagci et al. 2020". However, this
      annotation conflicts with multiple lines of evidence showing HINT2 has an
      N-terminal mitochondrial targeting sequence (aa 1-17 per UniProt) that directs
      the mature protein to the matrix. The Reactome interpretation may be erroneous
      or conflating HINT2 with a different protein.
    action: REMOVE
    reason: This annotation should be removed for several reasons. (1) HINT2 has a
      well-characterized N-terminal mitochondrial targeting sequence (aa 1-17 per
      UniProt FT TRANSIT) that directs the protein to the mitochondrial matrix, not
      the outer membrane. (2) The primary characterization study (PMID:16762638)
      localized HINT2 to mitochondria but did not demonstrate outer membrane
      localization specifically. (3) The Reactome citation to "Chen et al. 2017"
      for RHOD-HINT2 interaction triggering Ca2+ influx is questionable - more
      recent literature (e.g., PMC8677646) shows HINT2 interacts with MCU
      (mitochondrial calcium uniporter) which is in the inner membrane/matrix,
      not the outer membrane. The outer membrane localization may be a curation
      error conflating the Ca2+ signaling function with outer membrane localization.
      HINT2's N-terminal targeting sequence is incompatible with outer membrane
      localization of the mature protein.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
    - reference_id: Reactome:R-HSA-9693214
      supporting_text: GTP-bound RHOD also binds HINT2 at the mitochondrial outer
        membrane, which triggers mitochondrial Ca2+ influx
- term:
    id: GO:0016787
    label: hydrolase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for hydrolase activity based on phylogenetic inference
      across the HIT family. This is correct but overly general. HINT2 has specifically
      characterized adenosine 5'-monophosphoramidase activity (GO:0043530) which is
      a more informative term.
    action: MODIFY
    reason: While hydrolase activity is not incorrect, the more specific term GO:0043530
      (adenosine 5'-monophosphoramidase activity) is experimentally established with
      detailed kinetics (kcat = 0.0223 s-1, Km = 128 uM) and should be preferred.
    proposed_replacement_terms:
    - id: GO:0043530
      label: adenosine 5'-monophosphoramidase activity
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
    - reference_id: PMID:31990367
      supporting_text: Here, a similar substrate specificity profile (kcat /Km ) for
        model phosphoramidate substrates was found for hHINT2 but with higher kcat
        and Km values when compared with hHINT1
- term:
    id: GO:0000166
    label: nucleotide binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for nucleotide binding based on UniProt keyword mapping.
      This is true but uninformative for a nucleotide-processing enzyme. The nucleotide
      (AMP) is a product of the enzymatic reaction, not a regulatory binding site.
    action: MARK_AS_OVER_ANNOTATED
    reason: While HINT2 does bind nucleotides as part of its catalytic mechanism,
      this is inherent to its enzymatic function. The term "nucleotide binding" alone
      is uninformative and the more specific catalytic function (GO:0043530) better
      captures the biological role.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group
- term:
    id: GO:0003824
    label: catalytic activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: IEA annotation for generic catalytic activity based on InterPro domain
      mapping (HIT-like domain IPR011146). This is correct but extremely vague. The
      specific enzymatic activity (adenosine 5'-monophosphoramidase activity) is well-characterized.
    action: MODIFY
    reason: Generic "catalytic activity" should be replaced with the specific enzymatic
      function GO:0043530 (adenosine 5'-monophosphoramidase activity) which is experimentally
      validated.
    proposed_replacement_terms:
    - id: GO:0043530
      label: adenosine 5'-monophosphoramidase activity
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
- term:
    id: GO:0016787
    label: hydrolase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for hydrolase activity based on UniProt keyword mapping.
      This is a duplicate of the concept captured by the IBA annotation. The term
      is correct but too general.
    action: MODIFY
    reason: Should use the more specific GO:0043530 (adenosine 5'-monophosphoramidase
      activity) which is experimentally established.
    proposed_replacement_terms:
    - id: GO:0043530
      label: adenosine 5'-monophosphoramidase activity
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
- term:
    id: GO:0043530
    label: adenosine 5'-monophosphoramidase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: IEA annotation for the specific adenosine 5'-monophosphoramidase activity
      based on automated annotation (ARBA rule and Rhea reaction). This correctly
      captures the primary enzymatic function of HINT2.
    action: ACCEPT
    reason: Correct specific annotation for HINT2's primary molecular function. This
      is the core enzymatic activity with well-characterized kinetics (kcat = 0.0223
      s-1, Km = 128 uM for AMP-pNA substrate).
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:35156780
  review:
    summary: IPI annotation for protein binding with CFTR based on high-throughput
      mammalian membrane two-hybrid (MaMTH-HTS) screening. HINT2 is a mitochondrial
      matrix/inner membrane protein, while CFTR is a plasma membrane chloride channel.
      The physiological relevance of this interaction is highly questionable given
      the distinct subcellular compartments.
    action: REMOVE
    reason: This annotation should be removed because (1) "protein binding" is an
      uninformative term that does not specify the nature or consequence of the
      interaction, (2) the interaction was detected in a high-throughput artificial
      system (membrane two-hybrid) that may not reflect physiological conditions,
      and critically (3) HINT2 and CFTR localize to completely different subcellular
      compartments (mitochondria vs. plasma membrane), making physiological
      interaction extremely unlikely. HINT2 has an N-terminal mitochondrial targeting
      sequence and multiple studies confirm its mitochondrial localization
      (PMID:16762638). CFTR is a plasma membrane chloride channel. There is no
      plausible biological context where these proteins would interact in vivo.
      High-throughput protein-protein interaction screens frequently produce
      false positives, and this appears to be one.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 was localized in mitochondria.
    - reference_id: PMID:35156780
      supporting_text: high-throughput screening variant of the Mammalian Membrane Two-Hybrid
        (MaMTH-HTS) to map the protein-protein interactions
- term:
    id: GO:0043530
    label: adenosine 5'-monophosphoramidase activity
  evidence_type: IDA
  original_reference_id: PMID:16762638
  review:
    summary: Primary experimental evidence from Martin et al. (2006) establishing
      HINT2 as an adenosine 5'-monophosphoramidase. Using the model substrate AMP-pNA,
      they determined kcat = 0.0223 s-1 and Km = 128 uM. The active site histidine
      (H149) was confirmed by mutagenesis - H149A mutation abolished enzymatic activity.
    action: ACCEPT
    reason: Gold standard experimental evidence defining HINT2's core enzymatic function.
      This is the primary molecular function of HINT2 with detailed kinetic characterization
      and active site validation.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino
        group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
- term:
    id: GO:0043530
    label: adenosine 5'-monophosphoramidase activity
  evidence_type: IDA
  original_reference_id: PMID:31990367
  review:
    summary: Experimental evidence from Strom et al. (2020) comparing substrate specificities
      between HINT1 and HINT2. They confirmed similar phosphoramidate hydrolysis activity
      for both enzymes, with HINT2 showing higher kcat and Km values compared to HINT1.
      Also demonstrated a broader pH range for maximum catalytic activity (pK1 = 6.76,
      pK2 = 8.41).
    action: ACCEPT
    reason: Confirmatory experimental evidence for HINT2's adenosine 5'-monophosphoramidase
      activity with additional characterization of pH dependence and substrate specificity.
    supported_by:
    - reference_id: PMID:31990367
      supporting_text: Here, a similar substrate specificity profile (kcat /Km ) for
        model phosphoramidate substrates was found for hHINT2 but with higher kcat
        and Km values when compared with hHINT1
- term:
    id: GO:0006629
    label: lipid metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for lipid metabolic process based on UniProt keyword mapping
      (KW-0443 Lipid metabolism). This is derived from correlative evidence in
      PMID:18653718 where HINT2 knockdown affected steroidogenic response in H295R
      adrenocortical cells. The connection to lipid metabolism is indirect and
      mechanistically unclear - the authors explicitly state the mechanism "still
      remains to determine at the molecular level."
    action: MARK_AS_OVER_ANNOTATED
    reason: The evidence linking HINT2 to lipid metabolism is phenotypic and
      correlative, not mechanistic. PMID:18653718 shows that HINT2 knockdown
      reduced steroidogenic response, but the authors acknowledge the mechanism
      is unknown. More recent work (Wang et al. 2025) shows HINT2 deficiency
      exacerbates hepatic steatosis through effects on mitochondrial NAD+/SIRT3
      pathways, but this represents an indirect consequence of disrupted
      mitochondrial function rather than direct involvement in lipid metabolic
      enzymes or pathways. HINT2's primary function is adenosine 5'-monophosphoramidase
      activity - there is no evidence it directly participates in lipid metabolic
      reactions. The term "lipid metabolic process" is too broad and implies
      direct involvement that is not supported.
    supported_by:
    - reference_id: PMID:18653718
      supporting_text: these data suggest that, in H295R cells, Hint2 is required for an
        optimal steroidogenic response, possibly because of a particular signalling
        function exerted within the mitochondria and that still remains to determine at
        the molecular level.
    - reference_id: file:human/HINT2/HINT2-deep-research-falcon.md
      supporting_text: HINT2 loss reduces mitochondrial NAD+ and SIRT3 activity; loss of
        Hint2 decreased mitochondrial NAD+ and SIRT3 activity, increased mitochondrial
        protein hyperacetylation, and exacerbated diet-induced steatosis
- term:
    id: GO:0006694
    label: steroid biosynthetic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for steroid biosynthesis based on UniProt keyword mapping
      (KW-0752). This is derived from PMID:18653718 which showed that HINT2 knockdown
      in adrenocortical H295R cells reduced pregnenolone secretion in response to
      angiotensin II, K+, forskolin, or hydroxycholesterol stimulation. However,
      the mechanism is explicitly stated as unknown, and the effect appears to be
      through mitochondrial calcium signaling and membrane potential rather than
      direct participation in steroidogenic enzymes.
    action: MARK_AS_OVER_ANNOTATED
    reason: The evidence from Lenglet et al. (2008) is correlative and the mechanism
      is explicitly unknown. The abstract states that HINT2's effects "could be related
      to its ability to maintain a favorable mitochondrial potential" and that the
      mechanism "still remains to determine at the molecular level." Importantly,
      HINT2 overexpression had "no effect" on steroidogenesis - only knockdown showed
      effects, suggesting HINT2 is not rate-limiting but rather maintains basal
      mitochondrial function required for optimal steroidogenic capacity. HINT2 is
      NOT a steroidogenic enzyme (like CYP11A1, HSD3B, etc.) - it is an adenosine
      5'-monophosphoramidase. The steroidogenic effects are likely secondary to
      disruption of mitochondrial calcium handling or membrane potential rather
      than direct involvement in steroid biosynthetic pathways. This represents
      classic over-annotation from phenotypic observations.
    supported_by:
    - reference_id: PMID:18653718
      supporting_text: these data suggest that, in H295R cells, Hint2 is required for an
        optimal steroidogenic response, possibly because of a particular signalling
        function exerted within the mitochondria and that still remains to determine at
        the molecular level.
    - reference_id: PMID:18653718
      supporting_text: Hint2 overexpression in H295R cells had no effect on pregnenolone
        secretion elicited by angiotensin II or K+, whereas protein silencing with
        specific small interfering RNA resulted in a marked reduction of the steroidogenic
        response.
    - reference_id: PMID:18653718
      supporting_text: Calcium-dependent and calcium-independent actions of Hint2 on
        steroidogenesis could be related to its ability to maintain a favorable
        mitochondrial potential.
- term:
    id: GO:0006915
    label: apoptotic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for apoptotic process based on UniProt keyword mapping
      (KW-0053). The original evidence from PMID:16762638 shows HINT2 overexpression
      sensitizes hepatoma cells to Fas-mediated apoptosis (reduced viability, enhanced
      mitochondrial depolarization, increased caspase cleavage). However, this is
      a PHENOTYPIC observation in a specific cell model, not evidence of direct
      mechanistic involvement in the apoptotic machinery. The authors describe HINT2
      as an "apoptotic sensitizer" - meaning it modulates sensitivity to apoptotic
      stimuli, likely through effects on mitochondrial function (membrane potential,
      NAD+ homeostasis), rather than being a core component of apoptotic signaling.
    action: MARK_AS_OVER_ANNOTATED
    reason: The evidence from Martin et al. (2006) demonstrates that HINT2 modulates
      cellular sensitivity to apoptotic stress, but does not establish HINT2 as a
      direct participant in apoptotic signaling pathways. The observed effects
      (mitochondrial depolarization, caspase activation) are downstream consequences
      of HINT2's role in mitochondrial function, not evidence of direct apoptotic
      function. The abstract explicitly states the "biological function" of HINT2
      is "uncertain" and classifies it as an "apoptotic sensitizer" rather than
      an apoptotic regulator. More recent studies (Wang et al. 2025, Fan et al. 2020)
      link HINT2 to mitochondrial NAD+ homeostasis and SIRT3-dependent deacetylation,
      suggesting the apoptotic phenotype is secondary to bioenergetic roles. GO
      annotation for "apoptotic process" implies direct involvement, which is not
      supported by the mechanistic evidence.
    supported_by:
    - reference_id: PMID:16762638
      supporting_text: Exposed to apoptotic stress, fewer HepG2 cells overexpressing
        Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed
        changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/-
        1.6%) with more cleaved caspases than control cells.
    - reference_id: PMID:16762638
      supporting_text: Hint2 defines a novel class of mitochondrial apoptotic sensitizers
        down-regulated in hepatocellular carcinoma.
    - reference_id: file:human/HINT2/HINT2-deep-research-falcon.md
      supporting_text: HINT2 modulates mitochondrial NAD+ homeostasis and promotes
        SIRT3-dependent deacetylation; overexpression increases mitochondrial NAD+
        and reduces mitochondrial protein hyperacetylation
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000024
  title: Manual transfer of experimentally-verified manual GO annotation data to orthologs
    by curator judgment of sequence similarity
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000107
  title: Automatic transfer of experimentally verified manual GO annotation data to
    orthologs using Ensembl Compara
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:16762638
  title: Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular
    carcinoma.
  findings:
  - statement: HINT2 is localized to mitochondria
    supporting_text: Hint2 was localized in mitochondria.
  - statement: Exhibits adenosine 5'-monophosphoramidase activity
    supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino group
      (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
  - statement: Catalysis depends on conserved histidine triad
    supporting_text: Hints, histidine triad nucleotide-binding proteins, are adenosine
      monophosphate-lysine hydrolases
  - statement: Functions as a mitochondrial apoptotic sensitizer
    supporting_text: Exposed to apoptotic stress, fewer HepG2 cells overexpressing
      Hint2 remained viable
  - statement: Highly expressed in liver and pancreas
    supporting_text: Hint2 was predominantly expressed in liver and pancreas
  - statement: Downregulated in hepatocellular carcinoma
    supporting_text: HINT2 messenger RNA is downregulated in hepatocellular carcinomas
- id: PMID:18653718
  title: Hint2 is expressed in the mitochondria of H295R cells and is involved in
    steroidogenesis.
  findings:
  - statement: HINT2 is expressed in mitochondria of adrenocortical H295R cells
    supporting_text: Hint2 is expressed in the mitochondria of H295R cells
  - statement: HINT2 is involved in steroidogenesis
    supporting_text: this protein is involved in steroidogenesis
- id: PMID:31990367
  title: Histidine triad nucleotide-binding proteins HINT1 and HINT2 share similar
    substrate specificities and little affinity for the signaling dinucleotide Ap4A.
  findings:
  - statement: HINT1 and HINT2 have similar substrate specificity profiles
    supporting_text: Here, a similar substrate specificity profile (kcat /Km ) for
      model phosphoramidate substrates was found for hHINT2 but with higher kcat and
      Km values when compared with hHINT1
  - statement: HINT2 has higher kcat and Km values compared to HINT1
    supporting_text: with higher kcat and Km values when compared with hHINT1
  - statement: HINT2 has a broader pH range for maximum catalytic activity
    supporting_text: A broader pH range for maximum catalytic activity was determined
      for hHINT2 (pK1  = 6.76 ± 0.16, pK2  = 8.41 ± 0.07)
  - statement: Neither HINT1 nor HINT2 bind Ap4A
    supporting_text: Ap4 A was found to have no detectable binding to HINT1 nor HINT2
      by isothermal titration calorimetry
- id: PMID:34800366
  title: Quantitative high-confidence human mitochondrial proteome and its dynamics
    in cellular context.
  findings:
  - statement: HINT2 identified in high-confidence human mitochondrial proteome by
      quantitative proteomics
    supporting_text: mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP)
- id: PMID:35156780
  title: CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput
    screening system.
  findings:
  - statement: HINT2 detected as CFTR interactor in high-throughput screening (physiological
      relevance unclear)
    supporting_text: high-throughput screening variant of the Mammalian Membrane Two-Hybrid
      (MaMTH-HTS) to map the protein-protein interactions
- id: Reactome:R-HSA-9693214
  title: RHOD binds effectors at the mitochondrial outer membrane
  findings:
  - statement: GTP-bound RHOD binds HINT2 at mitochondrial outer membrane
    supporting_text: GTP-bound RHOD also binds HINT2 at the mitochondrial outer membrane
  - statement: This interaction triggers mitochondrial Ca2+ influx
    supporting_text: which triggers mitochondrial Ca2+ influx
- id: file:human/HINT2/HINT2-deep-research-falcon.md
  title: Deep research summary for HINT2 functional annotation
  findings:
  - statement: HINT2 regulates mitochondrial NAD+ homeostasis and SIRT3-dependent
      deacetylation
  - statement: HINT2 overexpression is cardioprotective after myocardial infarction
  - statement: HINT2 is downregulated in hepatocellular carcinoma and colorectal cancer
core_functions:
- description: Adenosine 5'-monophosphoramidase activity - hydrolyzes purine nucleotide
    phosphoramidates with a single phosphate group (e.g., AMP-NH2 to AMP + NH2). Catalytic
    activity depends on the conserved His-X-His-X-His histidine triad motif, with
    His-149 essential for catalysis (kcat = 0.0223 s-1, Km = 128 uM for AMP-pNA substrate).
  molecular_function:
    id: GO:0043530
    label: adenosine 5'-monophosphoramidase activity
  locations:
  - id: GO:0005739
    label: mitochondrion
  supported_by:
  - reference_id: PMID:16762638
    supporting_text: Hint2 hydrolyzed adenosine monophosphate linked to an amino group
      (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L)
  - reference_id: PMID:31990367
    supporting_text: Here, a similar substrate specificity profile (kcat /Km ) for
      model phosphoramidate substrates was found for hHINT2
proposed_new_terms: []
suggested_questions:
- question: What is the physiological substrate of HINT2 in mitochondria?
- question: How does HINT2 regulate mitochondrial NAD+ homeostasis?
- question: Is HINT2 truly localized to the mitochondrial outer membrane in addition
    to the matrix?
suggested_experiments:
- description: Determine sub-mitochondrial localization using protease protection
    assays to clarify whether HINT2 is in the matrix (as predicted by targeting sequence)
    or accessible at the outer membrane (as suggested by RHOD interaction data).
- description: Identify endogenous substrates using metabolomics by comparing metabolite
    profiles in HINT2 knockout vs wildtype mitochondria to identify accumulating phosphoramidates
    that may be endogenous substrates.

Gene Description

Existing Annotations Review

Core Functions

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

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