Biology subjects

Subramaniam, S.

Publications and source records attributed to Subramaniam, S..

68 records · Page 4Linked to original sources

bioRxiv2020

SUMO Modifies GβL and Mediates mTOR Signaling

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications, however, underlying mechanisms remains unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, G{beta}L, is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that G{beta}L is SUMOylated at lysine sites K86, K215, K245, K261 and K305. We found that SUMO depletion reduces mTOR-Raptor and mTOR-Rictor complex formation and diminishes nutrient-induced mTOR signaling. Furthermore, we found that reconstitution with WT G{beta}L but not SUMOylation defective KR mutant G{beta}L promote mTOR signaling in G{beta}L-depleted cells. Taken together, we report for the very first time that SUMO modifies G{beta}L, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

biochemistry↗

bioRxiv2020

The Mammalian Target of Rapamycin (mTOR) Kinase Mediates Haloperidol-Induced Cataleptic Behavior

The mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase protein complex (mTORC1 or mTORC2) that orchestrates diverse functions ranging from embryonic development to aging. However, its brain tissue-specific roles remain less explored. Here, we have identified that the depletion of the mTOR gene in the mice striatum completely prevented the extrapyramidal motor side-effects (catalepsy) induced by the dopamine 2 receptor (D2R) antagonist haloperidol, which is the most widely used typical antipsychotic drug. Conversely, a lack of striatal mTOR in mice did not affect catalepsy triggered by the dopamine 1 receptor (D1R) antagonist SCH23390. Along with the lack of cataleptic effects, the administration of haloperidol in mTOR mutants failed to increase striatal phosphorylation levels of ribosomal protein pS6 (S235/236) as seen in control animals. To confirm the observations of the genetic approach, we used a pharmacological method and determined that the mTORC1 inhibitor rapamycin has a profound influence upon post-synaptic D2R-dependent functions. We consistently found that pretreatment with rapamycin entirely prevented (in a time-dependent manner) the haloperidol-induced catalepsy in wild-type mice. Collectively, our data indicate that striatal mTORC1 blockade may offer therapeutic benefits with regard to the prevention of D2R-dependent extrapyramidal motor side-effects of haloperidol in psychiatric illness.

neuroscience↗

bioRxiv2020

Rhes, a Striatal Enriched Protein, Regulates Post-Translational Small-Ubiquitin-like-Modifier (SUMO) Modification of Nuclear Proteins and Alters Gene Expression

Rhes (Ras homolog enriched in the striatum) is a multifunctional protein that orchestrates striatal toxicity, motor behaviors and abnormal movements associated with dopaminergic signaling, Huntington disease and Parkinson disease signaling in the striatum. Rhes engineers membranous tunneling nanotube-like structures and promotes intercellular protein and cargoes transport. Recent study revealed Rhes also regulates mitophagy via the Nix receptor. Despite these studies, the mechanisms through which Rhes mediates these diverse functions remains unclear. Rhes belongs to a small GTPase family member and consists of a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes the post-translational modification (PTM) of proteins with SUMO (SUMOylation) by promoting "cross-SUMOylation" of SUMO enzymes SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). However, the identity of the SUMO substrates of Rhes remains largely unknown. By combining high throughput interactome and SUMO proteomics we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene transcription. While Rhes has increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, it had decreased the SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 5 different lysine residues (K32, K110, K114, K120, K124 and K245). Furthermore, we found that Rhes regulates the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide a previously undescribed role for Rhes in regulating SUMOylation of nuclear targets and in orchestrating striatal gene expression via the SUMOylation.

biochemistry↗

bioRxiv2020

Plasmin cascade mediates thrombolytic events in SARS-CoV-2 infection via complement and platelet-activating systems

Recently emerged beta-coronavirus, SARS-CoV-2 has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age1-3. Recent clinical reports suggested that the pulmonary failure seen in COVID-19 may not be solely driven by acute ARDS, but also microvascular thrombotic events, likely driven by complement activation4,5. However, it is not fully understood how the SARS-CoV-2 infection mechanisms mediate thrombotic events, and whether such mechanisms and responses are unique to SARS-CoV-2 infection, compared to other respiratory infections. We address these questions here, in the context of normal lung epithelia, in vitro and in vivo, using publicly available data. Our results indicate that plasmin is a crucial mediator which primes interactions between complement and platelet-activating systems in lung epithelia upon SARS-CoV-2 infection, with a potential for therapeutic intervention.

immunology↗

bioRxiv2020

Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Parallel to Clathrin-mediated endocytosis, additional Clathrin-independent endocytic routes exist, including fast Endophilin-mediated endocytosis (FEME). The latter is not constitutively active but requires the activation of selected receptors. In cell culture, however, the high levels of growth factors in the regular culture media induce spontaneous FEME, which can be suppressed upon serum starvation. Thus, we predicted a role for protein kinases in this growth factor receptor-mediated regulation of the pathway. Using chemical and genetic inhibition, we found that Cdk5 and GSK3{beta} are negative regulators of FEME. Their inhibition was sufficient to activate FEME promptly in resting cells and boosted the production of endocytic carriers containing {beta} 1-adrenergic receptor, following dobutamine addition. We established that the kinases suppress FEME at several levels. They control Dynamin-1 and Dynein recruitment and sorting of cargo receptors such as Plexin A1 and ROBO1 into FEME carriers. They do so by antagonizing the binding of Endophilin to Dynamin-1 as well as to Collapsin response mediator protein 4 (CRMP4), a Plexin A1 adaptor. Cdk5 and GSK3{beta} also hamper the binding and recruitment of Dynein onto FEME carriers by Bin1. Interestingly, we found that GSK3{beta} binds to Endophilin, thus imposing a local regulation of FEME. Collectively, these findings place the two kinases as key regulators of FEME, licensing cells for rapid uptake by the pathway only upon when Cdk5 and GSK3{beta} activity is low.

cell biology↗

bioRxiv2020

cGAS, a DNA Sensor, Promotes Inflammatory Responses in Huntington Disease

The genetic cause of Huntington disease (HD) is attributed to the N-terminal polyglutamine expansion of huntingtin (mHTT). mHTT, which is a ubiquitously expressed protein, induces noticeable damage to the striatum, which affects motor, psychiatric, and cognitive functions in HD individuals. Although inflammatory responses apparently precede striatal damage and an overall progression of HD, the molecular mechanisms at work remain unclear (1-6). In this study, we found that cyclic GMP-AMP synthase (cGAS), a DNA sensor, which regulates inflammation, autophagy, and cellular senescence (7-9), plays a critical role in the inflammatory responses of HD. Ribosome profiling analysis reveals that cGAS mRNA has a high ribosome occupancy at exon 1 and codon-specific pauses at positions 171 (CCG) and 172 (CGT) in HD cells, compared to the control, indicating an altered cGAS expression. Accordingly, cGAS protein levels and activity, as measured by phosphorylation of stimulator of interferon genes (STING) or TANK-binding kinase 1 (TBK1), are increased in HD striatal cells, mouse Q175HD striatum and human postmortem HD striatum, compared to the healthy control. Furthermore, cGAS-dependent inflammatory genes such as Cxcl10 and Ccl5 show enhanced ribosome occupancy at exon 3 and exon 1, respectively and are upregulated in HD cells. Depletion of cGAS via CRISPR/Cas-9 diminishes cGAS activity and decreases expression of inflammatory genes while suppressing the autophagy upregulation in HD cells. We additionally detected the presence of numerous micronuclei, a known inducer of cGAS, in the cytoplasm of HD cells. Overall, the data indicates that cGAS is highly upregulated in HD and mediates inflammatory and autophagy responses. Thus, targeting cGAS may offer therapeutic benefits in HD.

neuroscience↗

bioRxiv2019

Biochemical and structural analysis reveals the Neurofibromin (NF1) protein forms a high-affinity dimer

Neurofibromin is the protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to aberrant signaling through the RAS-MAPK pathway due to disruption of the Neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of the majority of Neurofibromin--to date, biochemical and structural data exist only for the GAP domain and the region containing a Sec-PH motif. To better understand the role of this large protein, we carried out a series of biochemical and biophysical studies which demonstrate that full length Neurofibromin forms a high-affinity dimer. Neurofibromin dimerization also occurs in cells, and likely has biological and clinical implications. Analysis of purified full-length and truncated variants of Neurofibromin by negative stain electron microscopy reveals the overall architecture of the dimer and predicts the potential interactions which contribute to the dimer interface. Structures resembling high-affinity full-length dimers could be reconstituted by mixing N- and C-terminal protein domains in vitro. Taken together these data suggest how Neurofibromin dimers might form and be stabilized within the cell.

biochemistry↗

bioRxiv2019

RasGRP1 (CalDAG-GEF-II) Mediates L-DOPA-induced Dyskinesia in a Mouse Model of Parkinson Disease

The therapeutic benefits of L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson disease (PD) patients diminishes with the onset of abnormal involuntary movements (L-DOPA induced dyskinesia), a debilitating motor side effect. L-DOPA induced dyskinesia are due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions. However, the molecular mechanisms that promote L-DOPA-induced dyskinesia remain unclear. Here, we have reported that RasGRP1 (also known as CalDAG-GEF-II) physiologically mediated L-DOPA induced dyskinesia in a 6-hydroxy dopamine (6-OHDA) lesioned mouse model of PD. In this study, L-DOPA treatment rapidly upregulated RasGRP1 in the striatum. Our findings showed that RasGRP1 deleted mice (RasGRP1-/-) had drastically diminished L-DOPA-induced dyskinesia, and RasGRP1-/- mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediates L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway and binds directly with Ras-homolog-enriched in the brain (Rheb), which is a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase (Pde1c), Pde2a, catechol-o-methyltransferase (comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), which are downstream regulators of RasGRP1 and are linked to L-DOPA-induced dyskinesia vulnerability. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of L-DOPA-induced dyskinesia in the striatum. Drugs or gene-depletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing L-DOPA-induced dyskinesia in PD patients.

neuroscience↗

bioRxiv2019

Rhes, a Striatal-Enriched Protein, Promotes Mitophagy Via Nix.

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here we discovered that Rhes, a striatal-enriched protein, is a major regulator of mitophagy in the striatum. Rhes predominantly interact with dysfunctional mitochondria and degrades them via mitophagy, and this function is exacerbated by the striatal toxin, 3-nitropropionic acid (3-NP). 3-NP induces mitochondrial swelling, loss of cristae and neuronal cell death only in WT but not Rhes KO striatum. Mechanistically, Rhes disrupts the mitochondrial membrane potential ({Delta}{Psi}m) and interacts with mitophagy receptor, Nix. In Nix KO cells, Rhes fails to disrupt {Delta}{Psi}m or eliminate dysfunctional mitochondria. Moreover, Rhes travels to the neighboring cell and associates with dysfunctional mitochondria via Nix. Collectively, Rhes is a major regulator of mitophagy via Nix which may determine striatal vulnerability in the brain.

neuroscience↗

bioRxiv2019

Global ribosome profiling reveals that mutant huntingtin stalls ribosomes and represses protein synthesis independent of fragile X mental retardation protein

The regulators that stall ribosome translocation are poorly understood. We find that polyglutamine-expanded mutant Huntingtin (mHtt), the Huntingtons disease (HD) causing protein, promotes ribosome stalling and physiologically suppresses protein synthesis. A comprehensive, genome-wide analysis of ribosome footprint profiling (Ribo-Seq) revealed widespread ribosome stalling on mRNA transcripts and a shift in the distribution of ribosomes toward the 5 end, with single-codon unique pauses on selected mRNAs in HD cells. In Ribo-Seq, we found fragile X mental retardation protein (FMRP), a known regulator of ribosome stalling, translationally upregulated and it co-immunoprecipitated with mHtt in HD cells and postmortem brain. Depletion of FMRP gene, Fmr1, however, did not affect the mHtt-mediated suppression of protein synthesis or ribosome stalling in HD cells. Consistent with this, heterozygous deletion of Fmr1 in Q175FDN-Het mouse model, Q175FDN-Het; Fmr1+/-, showed no discernable phenotype, but a subtle deficit in motor skill learning. On the other hand, depletion of mHtt, which binds directly to ribosomes in an RNase-sensitive manner, enhanced global protein synthesis, increased ribosome translocation and decreased stalling. This mechanistic knowledge advances our understanding of the inhibitory role of mHtt in ribosome translocation and may lead to novel target(s) identification and therapeutic approaches that modulate ribosome stalling in HD.\n\nOne Sentence SummaryHuntingtons disease (HD) protein, mHtt, binds to ribosomes and affects their translocation and promotes stalling independent of the fragile X mental retardation protein.

neuroscience↗

bioRxiv2019

Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2 substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS ({omega}) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.

evolutionary biology↗

bioRxiv2019

Cryopreservation of Rosa hybrida cv. Helmut Schmidt by PVS2 vitrification method using in vitro fragmented explants (IFEs)

This is the first report on cryopreservation via PVS2 vitrification method on roses using in vitro fragmented explants (IFEs) as the starting material. The aim of this study is to optimize the efficient plant recovery and regeneration system for cryopreservation of Rosa hybrida cv. Helmut Schmidt using IFEs. Some important parameters have been optimized in this study are the effect of ascorbic acid (0.3 mM) examined separately and in combination at all steps in cryopreservation procedure (preculture, loading, unloading and growth recovery), loading type, loading duration, and PVS2 duration. The highest growth recovery of 43.33% was obtained when 3-4 mm size IFEs precultured on 0.25 M sucrose media supplemented with full-strength MS for one (1) day, followed by loading treatment supplemented with 1.5 M glycerol + 0.4 M sucrose + 5% DMSO + 0.3 mM ascorbic acid for 20 minutes, dehydration with PVS2 solution for 30 minutes and then treated with unloading solution supplemented with 1.2 M sucrose + 0.3 mM ascorbic acid for 20 minutes. This finding implies that long-term storage of Rosa Hybrida cv. Helmut Schmidt by PVS2 vitrification method was successful with essential biomolecules.

plant biology↗

bioRxiv2019

Dedifferentiation orchestrated through remodeling of the chromatin landscape defines PSEN1 mutation-induced Alzheimer's Disease

Early-Onset Familial Alzheimers Disease (EOFAD) is a dominantly inherited neurodegenerative disorder elicited by mutations in the PSEN1, PSEN2, and APP genes1. Hallmark pathological changes and symptoms observed, namely the accumulation of misfolded Amyloid-{beta} (A{beta}) in plaques and Tau aggregates in neurofibrillary tangles associated with memory loss and cognitive decline, are understood to be temporally accelerated manifestations of the more common sporadic Late-Onset Alzheimers Disease. The complete penetrance of EOFAD-causing mutations has allowed for experimental models which have proven integral to the overall understanding of AD2. However, the failure of pathology-targeting therapeutic development suggests that the formation of plaques and tangles may be symptomatic and not describe the etiology of the disease3,4. Here, we use an integrative, multi-omics approach and systems-level analysis in hiPSC-derived neurons to generate a mechanistic disease model for EOFAD. Using patient-specific cells from donors harboring mutations in PSEN1 differentiated into neurons, we characterize the disease-related gene expression and chromatin accessibility changes by RNA- Seq, ATAC-Seq, and histone methylation ChIP-Seq. We show that the defining disease-causing mechanism of EOFAD is dedifferentiation, primarily through the REST-mediated repression of neuronal lineage specification gene programs and the activation of non-specific germ layer precursor gene programs concomitant with modifications in chromatin accessibility. These gene signature profiles and changes in chromatin topology illustrate that EOFAD neurons traverse the chromatin landscape from an ectodermal origin to a mixed germ lineage state. Further, a reanalysis of existing transcriptomic data from PSEN1 patient brain samples demonstrates that the mechanisms identified in our experimental system recapitulate EOFAD in the human brain. Our results comprise a disease model which describes the mechanisms culminating in dedifferentiation that precede amyloid and tau pathology formation and engender neurodegeneration.

neuroscience↗

bioRxiv2019

Automated methods for 3D Segmentation of Focused Ion Beam-Scanning Electron Microscopic Images

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1-2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and analysis are two critical problems that limit more extensive use of FIB-SEM technology. Here, we present a robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for automated data segmentation using machine learning, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.\n\nImpact StatementThe high-throughput, easy-to-use and versatile segmentation pipeline described in our manuscript will enable rapid, large-scale statistical analysis of sub-cellular structures in tissues.

cell biology↗