Biology subjects

Kandasamy, G.

Publications and source records attributed to Kandasamy, G..

4 recordsLinked to original sources

bioRxiv2026

Trypsin exhibits exopeptidase-like activity toward N-terminal arginine that biases proteomic analyses

Many proteomics protocols rely on enzymatic digestion of complex protein mixtures to generate peptides with predictable cleavage patterns for the mass spectrometry analysis. One of the most utilized enzymes, trypsin, is classically defined as a serine endopeptidase with high specificity for cleaving peptide bonds on the C-terminal side of internal lysine and arginine residues. Accordingly, trypsin is not expected to remove the N-terminal arginine, which may arise through posttranslational modification such as arginylation or by proteolysis exposing internal residues as the new N-termini. N-terminal arginine plays important biological roles, including functioning as an N-degron and modulating protein interactions/signaling through its positive charge. Curiously, prior mass spectrometry-based studies utilizing trypsin to identify proteins bearing N-terminal arginine have frequently reported low and inconsistent yields, suggesting potential systematic bias in current proteomic approaches. Here, we explored whether trypsin would affect the integrity of the N-terminal arginine. By using antibodies specifically recognizing N-terminal arginine of different peptides, and by using mass spectrometry peptide analysis, we show that trypsin can remove N-terminal arginine residues in an exopeptidase-like manner. This effect occurs across a range of digestion conditions consistent with standard proteomic workflows, on peptides or whole proteins, and depends on trypsin concentration, incubation time, and catalytic activity. In addition, we show that the alternative arginine-cleavage enzyme Arg-C can also affect N-terminal arginine in a sequence-dependent context. In contrast, Lys-C and LysargiNase do not exhibit such effects, providing suitable alternative digestion strategies. Together, these findings reveal an unappreciated enzymatic behavior of arginine-cleaving proteases and suggest that their widespread use may systematically compromise the detection of N-terminal arginine in proteomic studies.

biochemistry↗

bioRxiv2024

Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

Cell death regulation is essential for stress adaptation and/or signal response. Past studies have shown that eukaryotic cell death is mediated by an evolutionarily conserved enzyme, arginyltransferase1 (Ate1). The downregulation of Ate1, as seen in many types of cancer, prominently increases cellular tolerance to a variety of stressing conditions. Conversely, in yeast and mammalian cells, Ate1 is elevated under acute oxidative stress conditions and this change appears to be essential for triggering cell death. However, studies of Ate1 were conventionally focused on its function in inducing protein degradation via the N-end rule pathway in the cytosol, leading to an incomplete understanding of the role of Ate1 in cell death. Our recent investigation shows that Ate1 dually exists in the cytosol and mitochondria, the latter of which has an established role in cell death initiation. Here, by using budding yeast as a model organism, we found that mitochondrial translocation of Ate1 is promoted by the presence of oxidative stressors and is essential for inducing cell death with characteristics of apoptosis. Also, we found that Ate1-induced cell death is dependent on the formation of the mitochondrial permeability pore and at least partly dependent on the action of mitochondria-contained factors including the apoptosis-inducing factor, but is not directly dependent on mitochondrial electron transport chain activity or its derived reactive oxygen species (ROS). Furthermore, our evidence suggests that, contrary to widespread assumptions, the cytosolic protein degradation pathways including ubiquitin-proteasome, autophagy, or endoplasmic reticulum (ER) stress response has little or negligible impacts on Ate1-induced cell death. We conclude that Ate1 controls the mitochondria-dependent cell death pathway.

biochemistry↗

bioRxiv2020

CCR4-NOT complex nuclease Caf1 is a novel shuttle factor involved in the degradation of ubiquitin-modified proteins by 26S proteasome

Protein degradation by ubiquitin proteasome system (UPS) is the major selective proteolytic pathway responsible for the degradation of short lived proteins ranging from regulatory proteins to abnormal proteins. Many diseases are associated with abnormal protein degradation; occasionally such dysregulated protein degradation is compensated by various transcriptional and translational control mechanisms in the cell. Among those pathways CCR4-NOT protein complex is responsible for transcriptional and transitional control of various gene expressions. Furthermore, CCR4-NOT complex also has a RING type ubiquitin ligase (E3) which is required for the degradation of several proteins. Here we report a novel function that the CCR4-NOT complex 3-5 exonuclease Caf1 is involved in ubiquitindependent degradation of short lived proteins by the 26S proteasome in yeast Saccharomyces cerevisiae. caf1 deletion results in stabilization of R-Ura3 (N-end rule) and Ub-V76-Ura3 (Ubiquitin fusion degradation) substrates from proteasomal degradation. Additionally, caf1 deletion accumulates ubiquitin-modified Ub-V76-Ura3 proteins and Caf1 binds to poly-ubiquitin conjugates and linear tetra ubiquitin chains. Surprisingly, Caf1 interacts with 19S regulatory particle complex of the 26S proteasome. Therefore, we conclude that Caf1 has an exciting novel function as an ubiquitin shuttle factor in which Caf1 targets ubiquitin-modified proteins to 26S proteasome for efficient degradation.

molecular biology↗

bioRxiv2020

Ccr4 is a novel shuttle factor required for ubiquitin-dependent protein degradation by the 26S proteasome

Degradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

genetics↗