Biology subjects

Negi, H.

Publications and source records attributed to Negi, H..

5 recordsLinked to original sources

bioRxiv2025

Structures of dynamic interactors at native proteasomes by PhIX-MS and cryoelectron microscopy

Proteasome function depends on a network of transient interactions that remain structurally and functionally unresolved. We developed PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry), a structural proteomics workflow that stabilizes transient interactions in cells by UV-activated crosslinking to capture topological information. Applying PhIX-MS with cryo-electron microscopy (cryo-EM), we mapped redox sensor TXNL1 at the proteasome regulatory particle (RP), placing its PITH domain above deubiquitinase RPN11 and resolving its dynamic thioredoxin domain near RPN2/PSMD1 and RPN13/ADRM1, ideally located to reduce substrates prior to proteolysis. We also resolved chaperone PSMD5 bound to RP without the proteolytic core particle (CP) where its C-terminus inserts into the ATPase pore blocking CP binding. PhIX-MS and AlphaFold modeling tether ubiquitin ligase UBE3C/Hul5 along the RP placing its catalytic site above the RPN11 active site, enabling their coupled activities. Our integrative approach enables the localization of native, low-affinity protein interactions and is broadly applicable to dynamic macromolecular assemblies.

cell biology↗

bioRxiv2022

Green leaf volatiles co-opt proteins involved in molecular pattern signaling in plant cells

The green leaf volatiles (GLVs) Z-3-hexen-1-ol and Z-3-hexenyl acetate are airborne infochemicals released from damaged plant tissues that prime defenses against herbivores and pathogens in receiver plants. They are conceptually similar to well-known damage-associated molecular patterns (DAMPs), but little is known about their mechanism of action. Using tomato cell cultures, we found that rapid responses to the two GLVs and the polypeptide DAMP systemin showed a significant overlap but also GLV-specific patterns. Within five minutes, GLVs induced changes in MAPK activity and proton-fluxes as well as rapid and massive changes in the phosphorylation status of proteins. Many of these proteins are involved in reprogramming the proteome from cellular homeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins, and transcriptional regulators, all of which are also components of DAMP signaling pathways. This phosphoproteome may represent an early priming state that enables plants to respond forcefully to a subsequent stress signal.

plant biology↗

bioRxiv2022

The proteasomal degradation signal FAT10 modulates substrate stability to accelerate degradation

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation activity remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags influences substrate degradation. The tag Fat10 is activated during inflammation, and ambiguously, Fat10 and its substrates undergo rapid proteasomal degradation. We report that the rapid degradation of Fat10 substrates depends on its structural plasticity. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate, and the mechanical unfolding kinetics of Fat10 regulates substrate degradation. Our studies reveal that long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Such a malleable structure also provides low resistance to mechanical unfolding and expedites proteasomal degradation. We also uncovered that the Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate. They correlated excellently to the regions where Fat10 contacts the substrate, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tag.

biochemistry↗

bioRxiv2019

A novel polyubiquitin chain linkage formed by viral Ubiquitin prevents cleavage by deubiquitinating enzymes

The Baculoviridae family of viruses encode a viral Ubiquitin gene. Although the viral Ubiquitin is homologous to eukaryotic Ubiquitin (Ub), preservation of this gene in the viral genome indicates a unique function that is absent in the host eukaryotic Ub. We report the structural, biophysical, and biochemical properties of the viral Ubiquitin from Autographa Californica Multiple Nucleo-Polyhedrosis Virus (AcMNPV). The structure of viral Ubiquitin (vUb) differs from Ub in the packing of the central helix 1 to the beta-sheet of the {beta}-grasp fold. Consequently, the stability of the fold is lower in vUb compared to Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54). The K54-linked polyubiquitin chains are neither effectively cleaved by deubiquitinating enzymes, nor are they targeted by proteasomal degradation. We propose that modification of proteins with the viral Ubiquitin is a mechanism to counter the host antiviral responses.

biochemistry↗

bioRxiv2019

The viral SUMO-targeted Ubiquitin Ligase ICP0 is phosphorylated and activated by host kinase Chk2

When the Herpes Simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called PML Nuclear Bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a SUMO-targeted Ubiquitin ligase ICP0 that degrades PML NBs to alleviate the repression. The molecular mechanism used by ICP0 to target PML NBs is unclear. For reasons unknown, the growth of HSV is dependent on the ATM/Chk2 pathway. Here we identify a bonafide SUMO-Interacting motif in ICP0 (SLS4) that is essential and sufficient to target SUMOylated proteins in PML NBs like PML and Sp100. Phosphorylation of SLS4 creates new salt-bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity and switches ICP0 into a potent STUbL. We also report that ICP0 exploits the kinase Chk2 to phosphorylate SLS4 and enhance its STUbL activity. Our results uncover how a viral STUbL counters antiviral response by exploiting an unprecedented mechanism involving three post-translational modifications; ubiquitination, SUMOylation, and phosphorylation.

biochemistry↗