Biology subjects

Hodul, M.

Publications and source records attributed to Hodul, M..

4 recordsLinked to original sources

bioRxiv2026

The WD40 domain of autophagy protein ATG-16.2 is required for lysosomal regulation and longevity in germline-less C. elegans

Autophagy is a conserved cellular recycling pathway linked to longevity and regulated by autophagy-related (ATG) protein. Many of these proteins serve a key function in ATG8 lipidation onto autophagosomal membranes, a step that precedes subsequent lysosomal degradation. Recently, mammalian ATG16L1, a member of ATG8 lipidation machinery, have been shown to also direct ATG8 lipidation onto other membranes via its C-terminal WD40 domain in acutely stressed cells. However, whether such non-canonical functions of the ATG16L1 WD40 domain influence longevity remain largely unknown. Here, we endogenously deleted the WD40 domain of atg-16.2, the C. elegans ortholog of ATG16L1, and examined autophagy, healthspan and longevity. The ATG-16.2 WD40 domain was dispensable for healthspan measures and for ATG8 lipidation in whole animals and neurons but was required for autophagic flux across all major tissues examined. Notably, the ATG-16.2 WD40 domain was also critical in limiting lysosomal membrane damage after acute heat stress. Because long-lived C. elegans mutants are typically heat resistant, we asked whether this lysosomal function supported their thermotolerance and found the WD40 domain to be selectively required for thermotolerance induced by germline removal, but not by reduced insulin/IGF-1 signaling or dietary restriction. In germline-less animals, loss of the ATG-16.2 WD40 domain reduced nuclear TFEB/HLH-30, reduced neuronal autophagic degradation, altered neuronal lysosomal pH, and sensitized animals to heat-induced lysosomal damage. Collectively, these findings establish the ATG16 WD40 domain as a regulator of autophagic degradation and lysosomal integrity in vivo, and show that this role is associated with longevity in a pathway-selective manner.

genetics↗

bioRxiv2024

Prosaposin is cleaved into saposins by multiple cathepsins in a progranulin-regulated fashion

Prosaposin (PSAP) is a lysosomal protein that plays a key role in sphingolipid metabolism. PSAP is cleaved into four bioactive disulfide-rich peptides, saposins A, B, C, and D, that catalyze sphin-golipidases to promote sphingolipid breakdown. Considering the key role of PSAP and saposins in sphingolipid metabolism and the existence of genetic mutations in PSAP associated with juve-nile-onset lysosomal storage and adult-onset neurodegenerative diseases, maintaining optimal levels of PSAP and saposins is crucial for proper lysosomal function and sphingolipid homeosta-sis. Despite this, the mechanism by which saposins are released from PSAP, and thus available to modulate sphingolipidases, sphingolipid homeostasis, and downstream lysosomal function, is not well understood. Here, we performed a comprehensive study to identify lysosomal enzymes which regulate prosaposin cleavage into saposins. In vitro cleavage assays identified multiple enzymes that can process human prosaposin into multi- and single-saposin fragments in a pH-dependent manner. We confirmed the role of cathepsins D and B in PSAP processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, K, L, S, V, G and AEP/LGMN) are able to process PSAP in distinctive, pH-dependent manners. In addition, we have demonstrated a novel role for progranulin (PGRN) in the regulation of PSAP cleavage. We found that PGRN and multi-granulin fragments (MGFs) directly regulate the cleavage of PSAP by cathepsin D. With this study, we have identified that multiple cathepsins, PGRN and MGFs work in concert to produce saposins under different conditions, which could present novel opportunities to modulate saposin levels in disease.

cell biology↗

bioRxiv2023

Interaction with the membrane-anchored protein CHIC2 constrains the ubiquitin ligase activity of CHIP

Maintenance of cellular health requires the proper regulation of E3 ubiquitin ligases. The E3 ligase CHIP is canonically regulated by its interactions with the molecular chaperones Hsp70 and Hsp90, which focus CHIPs ubiquitination activity on misfolded proteins. Here, we report a chaperone-independent interaction of CHIP with the membrane-anchored protein CHIC2, which strongly attenuates CHIPs ligase activity. We show that CHIC2 outcompetes abundant, cytosolic chaperones through its exquisite CHIP selectivity, rather than through enhanced affinity. In proteomic experiments, we find that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIPs functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.

biochemistry↗

bioRxiv2019

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, mechanisms that regulate the deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from target proteins are poorly understood. We previously showed that the DUB USP-46 removes ubiquitin from the glutamate receptor GLR-1 and regulates it trafficking and degradation in C. elegans. We found that WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identify another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome promotes the abundance of USP-46, and this effect is non-additive with increased expression of WDR-48. We found that USP-46 is ubiquitinated, and expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the half-life of USP-46. A point mutant version of WDR-48 that disrupts binding to USP-46 is unable to promote USP-46 abundance in vivo. Together, these data support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism to control DUB availability and function.

biochemistry↗