Biology subjects

Das, C.

Publications and source records attributed to Das, C..

5 recordsLinked to original sources

bioRxiv2026

Substrate biasing in UCHL5 proteoforms

Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.

biochemistry↗

bioRxiv2026

Identification, optimization, and structural elucidation of chloroacetamide scaffold as covalent inhibitors for Ubiquitin C-terminal Hydrolase L3

The deubiquitinating enzyme, ubiquitin C-terminal hydrolase L3 (UCHL3), has been implicated as a potential therapeutic target for cancer with a role in regulating the DNA damage response pathways. While the target has been studied using genetic methods there is a lack of reliable chemical probes to selectivity target UCHL3. In this study we report hit identification and optimization of a new chemical scaffold that irreversibly inhibits UCHL3. The observed structure-activity relationships are corroborated by ligand-bound crystal structures that confirm covalent adduct formation with the catalytic cysteine of the enzyme. Finally, through gel-shift assays using a ubiquitin activity-based probe we demonstrate on-target engagement with UCHL3 in two cell lines. The work as a whole presents a comprehensive evaluation of the new scaffold that can be utilized to probe UCHL3 in different biological contexts.

pharmacology and toxicology↗

bioRxiv2025

Dehydrating microhabitats increase mite activity and intensify ectoparasitism of Drosophila

Parasites interact with their host in variable environments that are often subject to water scarcity and dehydration. Drosophila fruit flies and associated ectoparasitic mites interact across a range of microhabitats, typically in decaying organic matter, such as fallen fruit and cactus tissue, that dries out and deteriorates over periods ranging from days to months. Here, we report that mite parasitism of Drosophila increases with exposure to increasingly dry conditions for two fly-mite systems (D. nigrospiracula-Macrocheles and D. melanogaster-Gamasodes). In D. melanogaster, artificial selection for behavioral resistance did not eliminate this effect, as previously selected lines remained relatively more resistant than non-selected controls even under dry conditions. Water balance assays confirmed that mites became dehydrated when held under dry conditions, which was also associated with increased mite activity. Exposure of D. melanogaster to mites dehydrated by exposure to low relative humidity increased parasitism, further supporting that mite infestation intensifies under dry conditions. The results indicate that ectoparasitism in this system is affected by the water content of the mites. The increased motivation of mites to parasitise flies under dry conditions may serve to replenish mite water stores and facilitate dispersal to more favorable microhabitats.

physiology↗

bioRxiv2025

Site-Specific Nanobody Inhibitors of the Proteasomal Deubiquitinase UCH37

Dysregulation of the ubiquitin (Ub) proteasome system (UPS) is linked to numerous human diseases, making its components attractive therapeutic targets. Among these, the proteasomal deubiquitinase UCH37 (also known as UCHL5) has two distinct Ub-processing activities: C-terminal Ub hydrolysis and K48-linkage-specific chain debranching. Separating these activities has not been possible with existing small-molecule inhibitors, which target the catalytic site and broadly affect both functions along with many other DUBs. Here, we report the development of two site-specific nanobody inhibitors of UCH37 that together enable this separation. Using yeast surface display, we generated Nb-cS1, which binds the canonical Ub-binding (S1) site and blocks both activities, and Nb-ncS1, which binds the non-canonical K48-specific (ncS1) site and selectively impairs chain debranching without affecting C-terminal Ub hydrolysis. X-ray crystallography and hydrogen-deuterium exchange mass spectrometry define the distinct binding surfaces engaged by each nanobody. In cells, both nanobodies selectively engage UCH37, with Nb-ncS1 showing the cleanest target engagement. These tools provide the first means to independently interrogate UCH37s two enzymatic activities and offer new approaches for dissecting the distinct roles of UCH37 in proteasomal and chromatin-associated contexts.

biochemistry↗

bioRxiv2020

Global Spread of SARS-CoV-2 Subtype with Spike Protein Mutation D614G is Shaped by Human Genomic Variations that Regulate Expression of TMPRSS2 and MX1 Genes

COVID-19 pandemic is a major human tragedy. Worldwide, SARS-CoV-2 has already infected over 3 million and has killed about 230,000 people. SARS-CoV-2 originated in China and, within three months, has evolved to an additional 10 subtypes. One particular subtype with a non-silent (Aspartate to Glycine) mutation at 614th position of the Spike protein (D614G) rapidly outcompeted other pre-existing subtypes, including the ancestral. We assessed that D614G mutation generates an additional serine protease (Elastase) cleavage site near the S1-S2 junction of the Spike protein. We also identified that a single nucleotide deletion (delC) at a known variant site (rs35074065) in a cis-eQTL of TMPRSS2, is extremely rare in East Asians but is common in Europeans and North Americans. The delC allele facilitates entry of the 614G subtype into host cells, thus accelerating the spread of 614G subtype in Europe and North America where the delC allele is common. The delC allele at the cis-eQTL locus rs35074065 of TMPRSS2 leads to overexpression of both TMPRSS2 and a nearby gene MX1. The cis-eQTL site, rs35074065 overlaps with a transcription factor binding site of an activator (IRF1) and a repressor (IRF2). IRF1 activator can bind to variant delC allele, but IRF2 repressor fails to bind. Thus, in an individual carrying the delC allele, there is only activation, but no repression. On viral entry, IRF1 mediated upregulation of MX1 leads to neutrophil infiltration and processing of 614G mutated Spike protein by neutrophil Elastase. The simultaneous processing of 614G spike protein by TMPRSS2 and Elastase serine proteases facilitates the entry of the 614G subtype into host cells. Thus, SARS-CoV-2, particularly the 614G subtype, has spread more easily and with higher frequency to Europe and North America where the delC allele regulating expression of TMPRSS2 and MX1 host proteins is common, but not to East Asia where this allele is rare.

genomics↗