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:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

GO_REF:0000117

Electronic Gene Ontology annotations created by ARBA machine learning models

PMID:15071506

A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier

UFM1 was identified as a novel ubiquitin-fold modifier with a dedicated conjugation system involving E1 (UBA5) and E2 (UFC1) enzymes, conserved in metazoa and plants but not yeast.

"Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast"

PMID:17182609

Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2

UfSP1 and UfSP2 were identified as cysteine proteases specific for UFM1, with UfSP2 conserved across multicellular organisms.

"UfSP2 is present in most, if not all, of multicellular organisms"
UfSP1/2 are strictly specific for UFM1 and do not cleave ubiquitin, SUMO-1, or ISG15.

"cleaved the C-terminal extension of Ufm1 but not that of ubiquitin"
The catalytic cysteine is essential for protease activity.

"replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities"

PMID:21228277

Structure of ubiquitin-fold modifier 1-specific protease UfSP2

Crystal structure of mouse UfSP2 reveals a two-domain architecture with an unconventional catalytic triad (Cys294, Asp418, His420).

"The C-terminal catalytic domain is similar to UfSP1 with Cys(294), Asp(418), His(420), Tyr(282), and a regulatory loop participating in catalysis"
The N-terminal domain of UfSP2 mediates substrate recognition and ER recruitment via interaction with DDRGK1 (C20orf116).

"The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum"

PMID:30626644

Ribosomal protein RPL26 is the principal target of UFMylation

RPL26 is the primary cellular UFMylation target, modified at K132/K134 on ER-bound 60S subunits. UFSP2 reverses this modification.

"a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation"
RPL26 UFMylation and deUFMylation occur at the ER surface via tethered enzyme complexes.

"RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER"
UFMylated RPL26 is enriched on ER-associated ribosomes.

"UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes"

PMID:37036982

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum

RPL26 UFMylation is required for degradation of stalled nascent chains at the ER via RQC and the ubiquitin-proteasome system.

"UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs"

PMID:26428751

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

A Y290H mutation in UFSP2 abolishes deUFMylase activity and causes autosomal dominant Beukes hip dysplasia.

"the BHD mutation abolishes UFSP2-mediated C-terminal cleavage of its substrate, Ufm1"

PMID:33473208

A pathogenic UFSP2 variant in an autosomal recessive form of pediatric neurodevelopmental anomalies and epilepsy

A homozygous V115E variant in the N-terminal domain of UFSP2 causes neurodevelopmental delay and epilepsy by impairing deUFMylation.

"the variant more prominently affects de-UFMylation rather than UFMylation"

PMID:38383785

UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER

UFMylation of uL24/RPL26 on ER-associated 60S ribosomal subunits facilitates rescue and recycling of the large subunit after ribosome-associated quality control splits stalled ribosomes during co-translational translocation at the ER.

"UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER."

file:DANRE/ufsp2/ufsp2-deep-research-bioreason-sft.md

BioReason SFT functional summary for ufsp2 (Danio rerio)

BioReason SFT correctly identifies ufsp2 as a cytoplasmic cysteine protease specializing in UFM1 processing and deconjugation, with a two-domain architecture supporting ER-proximal protein quality control.

file:DANRE/ufsp2/ufsp2-deep-research-falcon.md

Falcon deep research report on ufsp2 (Danio rerio)

Zebrafish ufsp2 (UniProt Q7T347) is the Ufm1-specific protease 2, a peptidase C78 family cysteine protease in the UFMylation system that acts as a UFM1-directed deconjugating enzyme and pro-UFM1 maturase, typically positioned at the endoplasmic reticulum through interaction with ODR4. No direct zebrafish primary studies were recovered, so function is inferred from conserved cross-species (human/mouse) mechanistic studies.

"The UniProt accession **Q7T347** corresponds to **Danio rerio ufsp2**, annotated as **Ufm1-specific protease 2 (UfSP2)**, a cysteine protease in the UFMylation (UFM1 conjugation) system. In the retrieved literature corpus, “UFSP2/Ufsp2” consistently refers to the **UFM1-specific protease 2** (not an unrelated gene), functioning as a UFM1-directed deconjugating enzyme and pro-UFM1 maturase, typically positioned at the **endoplasmic reticulum (ER)** through interaction with **ODR4**."
UFSP2 is a UFM1-specific cysteine protease/isopeptidase whose activity depends on a catalytic cysteine and a conserved Cys-Asp-His catalytic triad; activity is abolished by thiol-reactive reagents such as N-ethylmaleimide or by mutation of the catalytic cysteine.

"UFSP2 is described as a UFM1-specific **cysteine protease/isopeptidase**, with activity dependent on a catalytic cysteine and a conserved **Cys–Asp–His** catalytic triad (often discussed alongside an additional conserved Tyr typical of cysteine-based Ub/UBL proteases). UFSP activity can be inhibited by thiol-reactive reagents such as **N-ethylmaleimide (NEM)** or by mutation of the catalytic cysteine."
UFSP2 catalyzes two reactions: proteolytic maturation of pro-UFM1 to mature UFM1 required for UFMylation, and hydrolysis of isopeptide bonds linking UFM1 to substrate proteins (de-UFMylation). pro-UFM1 activation requires removal of a Ser84-Cys85 dipeptide to expose the conjugatable UFM1 C-terminus.

"Cross-species evidence supports that UFSP2 catalyzes: - **Proteolytic maturation of pro-UFM1** to mature UFM1 required for UFMylation, and - **Hydrolysis of isopeptide bonds** linking UFM1 to substrate proteins (de-UFMylation)."
A functional division of labor is described: UFSP2 is the major de-UFMylase for ER-associated substrates, efficiently removing UFM1 from ribosomal protein RPL26 (uL24); UFSP2 knockout causes drastic accumulation of UFMylated proteins. UFSP1 is more efficient for pro-UFM1 maturation and de-UFMylation of UFC1 at Lys122.

"- **UFSP2**: a major **de-UFMylase** for ER-associated substrates, particularly efficiently removing UFM1 from the ribosomal protein **RPL26 (uL24)**; UFSP2 knockout causes a **drastic accumulation of UFMylated proteins** in cell systems summarized in reviews."
UFSP2 can also reverse UFMylation on model substrates such as DDRGK1/UFBP1 and ASC1 in vitro and in cell studies, in addition to RPL26/uL24.

"UFSP2 can also reverse UFMylation on model substrates such as **DDRGK1/UFBP1** and **ASC1** in vitro/cell studies."
UFSP2 is positioned at the endoplasmic reticulum via interaction with the ER membrane protein ODR4, tethering it spatially to major UFMylation events on ER-associated ribosomes and thus to ER proteostasis and ribosome quality control. Reviews also list nucleus/cytoplasm/ER localization, but ER tethering is the strongest mechanistic conclusion.

"In the current pathway-centric view, this ER tethering spatially matches UFSP2 to major UFMylation events on **ER-associated ribosomes**, and thus to ER proteostasis/ribosome quality control processes."
A 2024 Nature study frames UFSP2 as an ER-tethered UFM1-specific hydrolase in a model where the UFMylation machinery promotes recycling of 60S ribosomal subunits from the ER, consistent with UFSP2's role in ribosome disassembly/recycling during ER-associated quality control.

"A 2024 Nature study (“UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER”) provides primary, high-authority support for a model in which UFMylation machinery promotes **recycling of 60S ribosomal subunits** from ER-associated states, and it explicitly frames UFSP2 as an **ER-tethered UFM1-specific hydrolase** in this functional context."