Biology subjects

Swarup, G.

Publications and source records attributed to Swarup, G..

8 recordsLinked to original sources

bioRxiv2024

Comparative vitreous proteomic profiling of proliferative diabetic retinopathy and diabetic with no-retinopathy subjects implicates impaired autophagy in DR pathogenesis

PurposeDiabetic retinopathy (DR) is a neurovascular complication of diabetes (DM) causing the loss of neurons (ganglion cells) in the retina. The disease etiology and potential pathogenic mechanisms in this disease remains unclear. In the present study, we aimed to further understand the key and novel pathogenic mechanisms involved in DR pathogenesis by taking cues from our global proteomics data. MethodologyThe study was approved by the institutional review board (IRB) of LVPEI, Hyderabad, India. Vitreous humour samples (PDR; n=3, DM; n=3, Control; n=3) were collected from patients undergoing vitrectomy and subjected to LC-MS-MS analysis. The acquired raw data were searched against the human vitreous proteome and was further analysed by various bioinformatic and proteomic tools. Western blotting and IHC was performed to validate crucial pathways. Blood samples from patients (DM, PDR & NPDR) and controls (n=50); cadaveric retinas from diabetic and non-diabetic donors (n=10) and epiretinal membranes (ERM, n=10) from DR cases and controls were collected and RNA was isolated. Quantitative expression of genes involved in autophagy were performed. CT was compared across different categories and significance estimated using a student t-test. ResultA total of 1079 proteins were identified with 16 completely novel proteins in eye proteome. Top pathways identified were autophagy, inflammation, LXR/RXR activation (lipid metabolism), ROS generation by macrophages, apoptosis and protein degradation. Regulatory proteins identified were associated with cell death, phagocytic activation, angiogenesis and apoptosis. Autophagy inducers such as ROS was found to be accumulated in the DR vitreous. TREM2, microglial receptor was identified as a novel protein in PDR vitreous. The expression of TREM2, an autophagy-associated gene was significantly (p-value = 0.05) upregulated in all categories as compared to control (NDM and/or NDM/No-DR). TREM2 protein also seemed to colocalise with microglial marker F4/80 in retinal tissues and intense expression was observed near the blood vessels in case of PDR retina. Other autophagy-associated markers were also differentially regulated in DR as compared to controls. ConclusionThis study emphasises on the strong role of autophagy pathways and its associated genes in the development of DR.

genomics↗

bioRxiv2023

Optineurin is involved in regulating macrophage responses during mycobacterial infection

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn+/+ and Optn-/- mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn-/- mice-derived macrophages showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the optineurin-deficient macrophages. Optineurin-deficient macrophages showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1{beta}. These observations taken together suggest a novel regulatory role for optineurin during mycobacterial infection, with its deficiency leading to an impairment in macrophage responses.

cell biology↗

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

Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3

NLRP3 is a cytoplasmic receptor protein, which initiates caspase-1 mediated inflammatory immune response upon detection of invading pathogen or a wide array of internal distress signals. Several gain-of function mutations of NLRP3 cause hereditary disorder of cold-induced hyper-inflammation known as familial cold autoinflammatory syndrome-1 (FCAS1). Although, caspase-1 activation and downstream interleukin-1{beta} / interleukin-18 maturation are common effectors in pathophysiology of this disorder, molecular mechanisms of how exposure to subnormal temperature triggers mutant NLRP3-inflammsome activity is not understood. Here, we show that endogenous NLRP3 is in complex with HSC70 (HSPA8), and this interaction is reduced upon exposure to cold. FCAS-causing NLRP3-L353P and NLRP3-R260W mutants show enhanced interaction with HSC70. Upon exposure to subnormal temperature, NLRP3-L353P and NLRP3-R260W show enhanced inflammasome formation, increased caspase-1 activation and reduced interaction with HSC70. Knockdown of HSC70 results in increased inflammasome formation by L353P and R260W mutants of NLRP3. Our results suggest that interaction with HSC70 suppresses inflammasome formation by FCAS-causing NLRP3 mutants at physiological temperature, and loss of this inhibitory association at subnormal temperature causes aggravated inflammasome formation and caspase-1 activation leading to interleukin-1{beta} maturation. These results provide evidence for HSC70 being a cold-sensor and a temperature-dependent regulator of inflammatory signaling by FCAS-causing NLRP3 mutants.

immunology↗

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

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↗

bioRxiv2019

HSC70 regulates cold-induced caspase-1 hyperactivation by an autoinflammation-causing mutant of cytoplasmic immune receptor NLRC4

NLRC4 is an innate immune receptor, which upon detection of certain pathogens or internal distress signal, initiates caspase-1 mediated inflammatory response. A gain-of-function mutation, H443P in NLRC4, causes familial cold autoinflammatory syndrome (FCAS) characterized by cold-induced hyperactivation of caspase-1 and inflammation. Here, we show that heat shock cognate protein 70 (HSC70) complexes with NLRC4 and negatively regulates caspase-1 activation by NLRC4-H443P. Compared to NLRC4, the structurally altered NLRC4-H443P shows enhanced interaction with HSC70. Knockdown of HSC70 or inhibition of its ATPase activity enhances caspase-1 activation by NLRC4-H443P. Exposure to subnormal temperature resulted in reduced interaction of NLRC4-H443P with HSC70, and an increase in its ability to form ASC-specks and activate caspase-1. By demonstrating that HSC70 differentially interacts with NLRC4-H443P mutant in a temperature-dependent manner to regulate caspase-1 activation, we provide a mechanism for cold-induced inflammation seen in FCAS patients with NLRC4-H443P mutation.

cell biology↗