Biology subjects

Moharir, S. C.

Publications and source records attributed to Moharir, S. C..

5 recordsLinked to original sources

bioRxiv2023

Transferrin-induced signaling through transferrin receptor and AKT kinase mediates formation of Rab8- and MICAL-L1-positive tubules involved in receptor recycling

Transferrin and its receptor play an important role in iron homeostasis. Binding of transferrin to its receptor (TFRC, transferrin receptor protein 1) initiates endocytic trafficking and subsequent recycling of TFRC to the plasma membrane. RAB8-positive tubules emanating from the endocytic recycling compartment play an important role in receptor recycling. However, the signaling pathways or mechanisms that mediate formation of RAB8-positive tubules are not clear. Here, we have investigated the role of transferrin-induced signaling in the regulation of RAB8- and MICAL-L1-positive tubules. Addition of transferrin to the serum starved HeLa cells resulted in enhanced recruitment of RAB8 as well as MICAL-L1 to the tubules, which was mediated by TFRC. Dynasore, an inhibitor of dynamin and endocytosis, completely blocked transferrin-induced formation of RAB8/MICAL-L1-positive tubules. RAB8 showed strong colocalization with MICAL-L1 on the tubules. Blocking of SRC or AKT kinase activity by specific inhibitors abolished transferrin-induced recruitment of RAB8 and MICAL-L1 to the tubules. Recycling of transferrin receptor was inhibited by blocking of AKT activity. TBC1D17, a GTPase activating protein for RAB8, inhibited RAB8/MICAL-L1-positive tubule formation. A phospho-mimicking mutant S366D of TBC1D17 did not inhibit formation of RAB8-positive tubules. Overall, these results show that transferrin induces TFRC mediated signaling dependent on endocytosis that is essential for the formation of RAB8- and MICAL-L1-positive tubules involved in recycling of transferrin receptor. Our results also show that AKT regulates transferrin-induced formation of RAB8- and MICAL-L1-positive tubules, which might be mediated by phosphorylation of TBC1D17.

cell biology↗

bioRxiv2022

Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/- MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

cell biology↗

bioRxiv2021

Optineurin deficiency induces patchy hair loss but it is not sufficient to cause amyotrophic lateral sclerosis in mice

Genetic alterations and environmental factors contribute towards pathogenesis of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Various types of mutations in the autophagy receptor protein optineurin (coded by OPTN gene) including deletions are causatively associated with ALS. To explore the role of OPTN in ALS pathogenesis, we used Optn knockout mice to study the features of ALS. The Optn-deficient mice did not show kyphosis, loss of body weight, weakening of front paw-grip strength or limb muscle strength. However, several Optn-deficient mice showed patchy loss of hair, which increased with age. Our results suggest that optineurin deficiency alone is not sufficient to induce ALS-like symptoms in mice. We suggest that optineurin deficiency may require cooperation with other genetic or environmental factors to cause ALS. Since endoplasmic reticulum (ER) stress plays an important role in ALS pathogenesis, and Optn modulates ER stress response signaling, Optn deficiency may contribute to ALS pathogenesis partly by potentiating ER stress response signaling.

neuroscience↗

bioRxiv2020

Overexpression of MutS impairs DNA mismatch repair and causes cell division defect in E.coli

MutS and its homologues, from prokaryotes to humans, recognize and bind to DNA mismatches generated during DNA replication, initiate DNA mismatch repair and ensures 100-200 fold increase in replication fidelity. In E.coli, through post transcriptional regulation, at least three mechanisms mediate decline of MutS intracellular concentrations during stress conditions. To understand the significance of this multifold regulation, we overexpressed MutS in E.coli and found that it led to impairment of DNA mismatch repair as reflected by preferential accumulation of transition mutations in spontaneous base pair substitution spectrum. This phenomenon was dependent on MutS-mismatch affinity and interaction. Higher MutS overexpression levels promoted DNA double strand breaks, inhibited cell division and resultantly caused a manifold increase in E.coli cell length. This cell division defect involved a novel MutS-FtsZ interaction and impediment of FtsZ ring function. Our findings may have relevance for cancers where mismatch proteins are known to be overexpressed.

genetics↗

bioRxiv2020

Optineurin promotes aggregation of mutant huntingtin and mutant ataxin-3, and reduces cytotoxicity of aggregates

Optineurin (OPTN), a cytoplasmic adaptor protein involved in cargo selective autophagy of bacteria, damaged mitochondria and mutant protein aggregates, is frequently seen in pathological structures containing protein aggregates, associated with several neurodegenerative diseases. However, the function of OPTN in these protein aggregates is not known. Here, we have explored the role of OPTN in mutant protein aggregation and in cytoprotection from toxicity of mutant proteins. Mutant huntingtin (mHtt) and mutant ataxin-3 (mAtax-3) showed reduced formation of aggregates in Optn-/- mouse embryonic fibroblasts as compared with wild type cells. Co-expression of OPTN enhanced aggregate formation by mHtt and mAtax-3 in Optn-/- cells. C-terminal domain of OPTN (412-577 amino acids) was necessary and sufficient to promote aggregate formation by these mutant proteins. The E478G mutant of OPTN, defective in ubiquitin-binding and autophagy, was also able to promote aggregation of mHtt and mAtax-3. OPTN and its C-terminal domain form a complex with the chaperone HSP70 known to promote mutant protein aggregation. Overexpression of mHtt or mAtax-3 induced more cell death in Optn-/- cells compared with wild type cells. Importantly, compared to wild type cells, Optn-deficient cells having mHtt or mAtax-3 aggregates showed higher level of cell death in neuronal (N2A) and non-neuronal cells. Our results show that OPTN promotes formation of mutant huntingtin and mutant ataxin-3 aggregates, and this function of OPTN might be mediated through interaction with HSP70 chaperones. Our results also show that OPTN reduces cytotoxicity caused by these mutant protein aggregates. Significance statementThe hallmark of several neurodegenerative diseases like amyotrophic lateral sclerosis, Huntingtons disease, Parkinsons disease, Alzheimers disease and Picks disease is the formation of pathological structures containing aggregated proteins, and OPTN is frequently observed in these structures. What role optineurin plays in those aggregates is not clear. Our results show that OPTN promotes aggregation of mutant huntingtin and mutant ataxin-3, and reduces cytotoxicity of aggregates in neuronal and non-neuronal cells. We suggest that OPTN provides cytoprotection in three different ways-by promoting mutant protein aggregation, by reducing cytotoxicity of aggregates and by autophagy-dependent clearance of aggregates reported earlier. These properties of OPTN provide a possible explanation for its association with various pathological structures containing protein aggregates seen in several neurodegenerative diseases.

neuroscience↗