miPEP155 (P155) is a 17-amino-acid human micropeptide translated from a 54-bp sense short open reading frame inside MIR155HG, the miR-155 host transcript (BIC), which is otherwise annotated as non-coding; it therefore has its own UniProt entry while sharing the host locus symbol. The peptide is made endogenously, as shown by a CRISPR knock-in EGFP reporter at the ORF stop codon, a peptide-specific antibody and mass spectrometry of immunoprecipitates, and the host transcript is strongly induced in antigen-presenting cells of inflamed skin. miPEP155 is found in the cytoplasm and nucleus and binds the nucleotide-binding (ATPase) domain, but not the substrate-binding domain, of the constitutive chaperone HSC70/HSPA8, lowering its ATP hydrolysis rate. In dendritic cells this weakens the HSC70-HSP90 co-chaperone machinery and HSC70's association with LAMP2A, so that internalised antigen is delivered less efficiently to lysosomes, surface MHC class II is reduced and priming of CD4+ T cells is dampened; synthetic peptide given to mice alleviates MOG-induced experimental autoimmune encephalomyelitis and imiquimod-induced psoriasis-like skin inflammation. The peptide does not regulate the level of its own primary miRNA transcript or the activity of miR-155, and appears functionally antagonistic to the microRNA it is co-transcribed with. Its coding sequence is well conserved in primates but is reported as absent or only partially conserved in mouse, whereas HSC70 itself is essentially identical between human and mouse.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0002605 negative regulation of dendritic cell antigen processing and presentation | IDA PMID:32671205 A micropeptide encoded by lncRNA MIR155HG suppresses autoimm... | ACCEPT | Summary: Synthetic P155 supplied to dendritic cells reduced delivery of soluble ovalbumin to LAMP2A-positive lysosomes, weakened the HSC70-HSP90 and HSC70-LAMP2A interactions, lowered MHC class II levels and impaired priming of OT-II CD4+ T cells. Direction (negative regulation) and cell type (dendritic cell) both match the evidence. Reason: This is the core biological role of the peptide and the term is at the right level of specificity. Two caveats should be read alongside it rather than against it: the trafficking, MHC class II and T cell priming readouts were obtained in mouse bone-marrow-derived dendritic cells treated with the human peptide (the authors state the coding sequence was not found in the mouse genome, and rely on human and mouse HSC70 being essentially identical), and all functional experiments used exogenously added synthetic peptide rather than loss of the endogenous peptide, which is difficult to achieve because the ORF lies within the miR-155 primary transcript. Target identification and the ATPase measurement were, however, done with human cells and recombinant human HSPA8, so the mechanism underlying this process term is human-grounded. Supporting Evidence: PMID:32671205 A weakened interplay between HSC70 and HSP90 as well as LAMP2A was detected in BMDCs cultured in the presence of P155 compared to BMDCs treated with Scr, which coincided with impaired lysosomal antigen trafficking observed in P155-treated BMDCs (Fig. PMID:32671205 We found that P155 treatment remarkably decreased MHC class II expression in BMDCs stimulated with R848, supporting the proposed model that P155 modulates antigen presentation (Fig. PMID:32671205 Although the coding sequences of P155 were not found in the mouse genome, we noticed that murine and human HSC70 share 99.85% homology (fig. |
| GO:0005634 nucleus | IDA PMID:32671205 A micropeptide encoded by lncRNA MIR155HG suppresses autoimm... | KEEP AS NON CORE | Summary: Immunofluorescence in HEK293T cells showed FITC-labelled synthetic peptide entering cells and colocalising with antibody-detected endogenous peptide in both cytoplasmic and nuclear compartments. Reason: The localisation observation is real and was made in human cells, so the annotation is retained. It is marked non-core because no nuclear activity has been demonstrated for the peptide: its only characterised partner, HSC70, is engaged in cytoplasmic and endolysosomal antigen trafficking, and the one proposed nuclear-type function for a miPEP, autoregulation of the host pri-miRNA, was tested and excluded for this peptide. Supporting Evidence: PMID:32671205 we first showed that fluorescein isothiocyanate (FITC)–labeled synthetic P155 efficiently entered HEK293T cells and colocalized with endogenous P155 in both cytoplasmic and nuclear compartments of the cells (fig. PMID:33810468 Thus, from our results we conclude that miPEP155 and miPEP497 do not regulate the levels of their own pri-miRNAs and consequently the activity of the processed miRNAs. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location keyword mapping (SL-0191 Nucleus), derived from the same imaging data as the IDA row above. Reason: The mapping is correct but adds nothing beyond the IDA row it restates, and the same reservation applies: nuclear residence is observed, nuclear function is not known. Supporting Evidence: PMID:32671205 we first showed that fluorescein isothiocyanate (FITC)–labeled synthetic P155 efficiently entered HEK293T cells and colocalized with endogenous P155 in both cytoplasmic and nuclear compartments of the cells (fig. |
| GO:0005737 cytoplasm | IDA PMID:32671205 A micropeptide encoded by lncRNA MIR155HG suppresses autoimm... | ACCEPT | Summary: Cytoplasmic localisation of the peptide shown by the same colocalisation experiment in HEK293T cells, and consistent with the compartment in which HSC70 operates during chaperone-mediated delivery of antigen to the lysosomal membrane. Reason: The cytoplasm is where the characterised molecular interaction takes place, so this is the functionally relevant location. The term is general, but for a 17-residue peptide with no membrane anchor and no reported organellar targeting signal there is no evidence supporting anything more specific. Supporting Evidence: PMID:32671205 we first showed that fluorescein isothiocyanate (FITC)–labeled synthetic P155 efficiently entered HEK293T cells and colocalized with endogenous P155 in both cytoplasmic and nuclear compartments of the cells (fig. PMID:32671205 Together with other cytoplasmic chaperones such as HSP90, HSC70 transports pentapeptide (KFERQ-like) motif–containing proteins delivered by CMA to the lysosomal membrane, facilitating MHC class II presentation of these antigens |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location keyword mapping (SL-0086 Cytoplasm), based on the same primary evidence as the IDA cytoplasm row. Reason: Correct mapping, concordant with the IDA row; redundant but not wrong. Supporting Evidence: PMID:32671205 we first showed that fluorescein isothiocyanate (FITC)–labeled synthetic P155 efficiently entered HEK293T cells and colocalized with endogenous P155 in both cytoplasmic and nuclear compartments of the cells (fig. |
| GO:0030544 Hsp70 protein binding | IPI PMID:32671205 A micropeptide encoded by lncRNA MIR155HG suppresses autoimm... | ACCEPT | Summary: Biotinylated peptide pulled a single ~73 kDa species out of human monocyte-derived dendritic cells, identified as HSC70/HSPA8 by mass spectrometry and immunoblot and competed away by free peptide; the interaction was reproduced in human THP-1-derived dendritic cells and mapped by pull-down and proximity ligation to the ATPase domain rather than the substrate-binding domain. Reason: This is an informative molecular function term, not bare protein binding, and names the right partner (P11142, HSPA8). The evidence is from human cells with a specific, competition-validated interaction and a mapped binding site. It understates the mechanism, however, since binding to the nucleotide-binding domain inhibits HSPA8 ATP hydrolysis; an ATPase inhibitor activity annotation is proposed separately rather than replacing this row, because the physical interaction and the inhibitory effect are separately evidenced and both are worth recording. Supporting Evidence: PMID:32671205 Using LC-MS and confirmative immunoblotting, we recognized this 73-kDa protein to be HSC70 (Fig. PMID:32671205 P155 did not bind to the general peptide-binding region, namely, the SBD domain of HSC70, but selectively bound to the ATPase region of HSC70 and suppressed the ATPase activity of full-length HSC70, which is critical for the HSC70-mediated chaperon cycle of peptide transportation (30). |
| GO:0042030 ATPase inhibitor activity | IDA PMID:32671205 A micropeptide encoded by lncRNA MIR155HG suppresses autoimm... | NEW | Summary: The peptide binds the nucleotide-binding (ATPase) domain of HSC70/HSPA8 and reduces the ATP hydrolysis activity of the full-length chaperone, measured with recombinant human HSPA8 and a scrambled peptide control. Reason: Proposed because this is the mechanistically informative molecular function and the one that makes the accepted process term intelligible, yet GOA records only the physical binding. The participation requirement is met at the molecular level: the peptide itself is the inhibitory entity acting directly on the enzyme, in a cell-free assay with purified human HSPA8, so this is not a necessity-versus-participation inference drawn from a perturbation phenotype. Comparator check (QuickGO, human, GO:0042030 exact): the small polypeptide ATPase inhibitors ATP5IF1 (IF1, ATP synthase) and PLN (phospholamban, SERCA2) carry this term by IDA, and FNIP1 and FNIP2, which inhibit the ATPase of the HSP90 chaperone, carry it by IDA too. Inhibitors of a chaperone ATPase are therefore annotated with this term rather than only a binding term, which is the convention followed here. Kept at the generic ATPase inhibitor level rather than inventing a chaperone-specific term, and asserted without a quantitative kinetic claim because the paper reports a colorimetric kit assay, not a full kinetic analysis. Supporting Evidence: PMID:32671205 Moreover, P155 remarkably impaired the ATPase activity of full-length HSC70, which functionally supported P155’s binding specificity (Fig. PMID:32671205 To determine the HSC70 ATPase activity, the recombinant protein of human heat shock 70-kDa protein 8 (HSPA8) transcript variant 1 (OriGene) was prepared in assay buffer at a final concentration of 0. |
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Download this section (compressed HTML)Q: Does the endogenous peptide, as opposed to synthetic peptide supplied at micromolar concentration, restrain antigen presentation? Every functional result for this entry comes from adding peptide from outside, and the experiment that would settle whether the annotations describe a physiological function or a pharmacological one has not been done.
Suggested experts: Hua Wang, Arnaud Besson
Q: Is the coding ORF genuinely absent from the mouse genome? The primary paper states it is and uses the human peptide on mouse cells for that reason, while a second group describes the peptide as partially conserved in mouse. The answer decides whether any mouse ortholog annotation is legitimate and whether mouse disease models are testing a conserved physiological axis or a human-peptide intervention.
Suggested experts: Jean-Philippe Combier, Serge Plaza
Q: Should the MHC class II route be annotated explicitly, with negative regulation of antigen processing and presentation of peptide antigen via MHC class II, alongside the dendritic-cell term already present? The mechanism runs through LAMP2A and surface MHC class II, but the two terms overlap heavily and the evidence comes from a single study, so this was left as a question rather than proposed.
Suggested experts: Hua Wang
Q: What does the peptide do in the nucleus? Nuclear residence is documented, host-pri-miRNA autoregulation has been excluded, and no nuclear partner is known.
Suggested experts: Arnaud Besson
Experiment: Disrupt the ORF without disturbing miR-155 production: introduce a start-codon or frameshift mutation in the sORF by base editing in human monocyte-derived or iPSC-derived dendritic cells, confirm by peptide-specific immunoblot and mass spectrometry that the peptide is lost while pri-miR-155 and mature miR-155 levels are unchanged, then measure antigen delivery to LAMP2A-positive compartments, surface MHC class II and autologous CD4+ T cell proliferation. The complementary control is to re-express the ORF from a heterologous promoter.
Hypothesis: The endogenous peptide, not just exogenously supplied peptide, limits MHC class II-restricted antigen presentation in human dendritic cells.
Experiment: Characterise the inhibition kinetically with purified human HSPA8: steady-state ATP hydrolysis across an ATP concentration series with and without peptide to establish the mode of inhibition and an IC50, isothermal titration calorimetry or surface plasmon resonance against the isolated nucleotide-binding domain to obtain an affinity, and a scrambled-sequence and alanine-scan series to identify the residues required. Pair this with a luciferase-refolding or client-release assay to show that the chaperone cycle itself, not only hydrolysis, is slowed.
Hypothesis: The peptide is a competitive inhibitor of nucleotide binding or of a nucleotide-cycle step of HSPA8, rather than an unspecific aggregation-prone peptide effect.
Experiment: In a non-immune human cell line, use a KFERQ-motif reporter to quantify chaperone-mediated autophagy flux with and without the peptide, and test whether the peptide alters HSPA8-LAMP2A co-immunoprecipitation and lysosomal translocation of a canonical substrate. A positive result would indicate that the dendritic-cell phenotype is one consequence of a broader effect on HSC70-dependent delivery, and would change which process terms are appropriate.
Hypothesis: The peptide acts on chaperone-mediated autophagy substrate delivery generally, not only on the dendritic-cell antigen route.
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