Biology subjects

Manifava, M.

Publications and source records attributed to Manifava, M..

4 recordsLinked to original sources

bioRxiv2026

Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration

Autophagy and mitophagy are essential cellular processes implicated in neurodegenerative diseases, yet few tractable targets have been robustly validated for therapeutic modulation of these pathways. In an industry-academia consortium we carried out an extensive literature review and expert curation, leading to selection of 29 genes previously reported to enhance autophagy upon genetic or pharmacological modulation. These genes were classified based on whether downregulation or overexpression induced autophagic activity. Using siRNA knockdown, small-molecule modulators, and transient transfection approaches, we systematically screened these targets in parallel across HeLa and HEK-293 cell lines using high- and low-content phenotypic imaging. Promising candidates were further evaluated in induced pluripotent stem cell (iPSC)-derived neurons and in Drosophila models. Three targets emerged as top candidates: HUWE1 downregulation consistently enhanced autophagy and aggregate clearance, while USP33 downregulation promoted mitophagy. We also identified USP20, a close homologue of USP33, as an inhibitor of mitophagy, a role not previously assigned to this protein. These effects on autophagy/mitophagy were corroborated in Drosophila, highlighting their potential functional relevance across species. This cross-platform, multi-laboratory study establishes a framework for identifying and validating regulators of autophagy and mitophagy and provides a route which enables a comprehensive reassessment of the current target literature in this field. The validation of HUWE1, USP20, and USP33 as candidate therapeutic targets offers new opportunities for intervention in neurodegenerative diseases.

neuroscience↗

bioRxiv2026

A GABARAP-PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation

Macroautophagy/autophagy is a cellular process enabling degradation of intracellular components during starvation. In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized within a brief time remains elusive. A protein complex central to the phagophore initiation is the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits a variety of downstream proteins, among which is PtdIns3P-binding WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that upon inhibition of mATG8 lipidation in cells, there is a decreased accumulation of WIPI2, suggesting a feedback loop between mATG8s and PtdIns3P production. The role of PtdIns3K-C1 in this feedback was demonstrated by in vitro experiments where recombinant membrane-coupled mATG8s bind to and potently activate PtdIns3K-C1, with GABARAP being the most potent activator among all mATG8s. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP binds two sites in PtdIns3K-C1, with one site showing an atypical bipartite interaction with the mATG8. We also confirm both sites are essential for GABARAP to activate PtdIns3K-C1. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be synthesized within a brief period. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/712327v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dc398eorg.highwire.dtl.DTLVardef@146a2cborg.highwire.dtl.DTLVardef@69c649org.highwire.dtl.DTLVardef@58fd81_HPS_FORMAT_FIGEXP M_FIG The GABARAP-PtdIns3K-C1 positive feedback loop. Model for the GABARAP-PtdIns3K-C1 positive feedback loop. GABARAP is indirectly recruited to the growing phagophore by PtdIns3P and activates PtdIns3K-C1, leading to an increased PtdIns3P production. The E1 (ATG7), E2 (ATG3) and E3 (ATG5-ATG12-ATG16L1) enzymes and WIPI2 are involved in the lipidation (covalent coupling) of GABARAP to membranes. C_FIG

molecular biology↗

bioRxiv2023

Ubiquitination is a novel post-translational modification of VMP1 in autophagy of human tumor cells

Autophagy is a tightly regulated catabolic process involved in the degradation and re-cycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin-RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor cell therapy resistance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/528037v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1a1538dorg.highwire.dtl.DTLVardef@74580corg.highwire.dtl.DTLVardef@409ef0org.highwire.dtl.DTLVardef@82b856_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Intracellular calcium elevations drive the nucleation of FIP200- and ATG13-containing pre-autophagosomal structures that become omegasomes

Ca2+ modulates autophagy at multiple steps including the induction and maturation of autophagosomes, but the magnitude and spatiotemporal properties of this calcium signal and its ultimate effect on the autophagic machinery are unclear. Focusing on the induction step leading to omegasome formation, we report that low but sustained elevations in cytosolic calcium levels induce omegasome formation but treatments that only transiently elevate calcium do not. The calcium-induced structures are early intermediates that mature into omegasomes but do not constitute full autophagosomes because they are partially devoid of late autophagy proteins ATG16 and LC3. In addition to omegasomes, all four components of the ULK complex (ULK1, FIP200, ATG13, ATG101) respond to calcium modulation: they translocate to early autophagy puncta in complete medium upon calcium elevation, and are inhibited from translocation during starvation by calcium chelation with BAPTA-2 AM. The principal early step affected by calcium lies downstream of mTORC1 inactivation and upstream of VPS34 activation, coinciding biochemically with phosphorylation of ATG13 at serine 318, which is known to require ULK1 activity. However, although the calcium-mediated step requires ATG9, FIP200 and ATG13, it does not require ULK1/2, suggesting that calcium does not directly regulate ULK1 activity but rather it regulates the mechanism by which the ULK complex components ATG13 and FIP200, together with ATG9, nucleate pre-autophagosomal precursors. This calcium-induced nucleation is sufficient to drive autophagy induction up to the omegasome step, but not beyond it.

cell biology↗