Biology subjects

Nagaraj, R.

Publications and source records attributed to Nagaraj, R..

8 recordsLinked to original sources

bioRxiv2024

Kingdom-specific lipid unsaturation shapes up sequence evolution in membrane arm subunits of eukaryotic respiratory complexes

Sequence evolution of protein complexes (PCs) is constrained by protein-protein interactions (PPIs). PPI-interfaces are predominantly conserved and hotspots for disease-related mutations. How lipid-protein interactions (LPIs) constrain sequence evolution of membrane- PCs? We explore Respiratory Complexes (RCs) as a case study as these allow to compare sequence evolution in subunits exposed to both lipid-rich inner-mitochondrial membrane (IMM) and aqueous matrix. We find that lipid-exposed surfaces of the IMM-subunits but not of the matrix subunits are populated with non-PPI disease-causing mutations signifying LPIs in stabilizing RCs. Further, IMM-subunits including their exposed surfaces show high intra- kingdom sequence conservation but remarkably diverge beyond. Molecular Dynamics simulation suggests contrasting LPIs of structurally superimposable but sequence-wise diverged IMM-exposed helices of Complex I (CI) subunit Ndufa1 from human and Arabidopsis depending on kingdom-specific unsaturation of cardiolipin fatty acyl chains. in cellulo assays consolidate inter-kingdom incompatibility of Ndufa1-helices due to the lipid- exposed amino acids. Plant-specific unsaturated fatty acids in human cells also trigger CI- instability. Taken together, we posit that altered LPIs calibrate sequence evolution at the IMM-arms of eukaryotic RCs.

cell biology↗

bioRxiv2023

Towards a human brain EV atlas: Characteristics of EVs from different brain regions, including small RNA and protein profiles

Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a "bdEV atlas" of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 for all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are merited to lend more insight into the links between EV heterogeneity and function in the CNS.

neuroscience↗

bioRxiv2023

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

genomics↗

bioRxiv2022

Surface phenotyping and quantitative proteomics reveal differentially enriched proteins of brain-derived extracellular vesicles in Parkinson's disease

Extracellular vesicles (EVs) are produced by all cell types and are found in all tissues and biofluids. EV proteins, nucleic acids, and lipids are a "nano-snapshot" of the parent cell that may be used for novel diagnostics of various diseases, including neurodegenerative disorders. Currently, diagnosis of the most common neurodegenerative movement disorder, Parkinsons disease (PD), relies on manifestations of late-stage progression, which may furthermore associate with other neurodegenerative diseases such as progressive supranuclear palsy (PSP). Here, we profiled surface markers and other protein contents of brain-derived extracellular vesicles (bd-EVs) from PD (n= 24), PSP (n=25) and control (n=24). bdEVs displayed tetraspanins and certain microglia, astrocyte, and neuron markers, while quantitative proteomics revealed enrichment of several proteins in PD vs. control and/or PSP, including clathrin heavy chain 1 and 14-3-3 protein gamma. This characterization of EVs in the source tissue provides insights into local dynamics as well as biomarker candidates for investigation in peripheral fluids.

neuroscience↗

bioRxiv2021

A proteomic perspective and involvement of cytokines in SARS-CoV-2 infection

Infection with the SARS-CoV-2 virus results in manifestation of several clinical observations from asymptomatic to multi-organ failure. Biochemically, the serious effects are due to what is described as cytokine storm. The initial infection region for COVID-19 is the nasopharyngeal/oropharyngeal region which is the site where samples are taken to examine the presence of virus. We have earlier shown that several defensin genes are down regulated in cells from this region in patients who tested positive in the RTPCR test. We have now carried out detailed proteomic analysis of the nasopharyngeal/oropharyngeal swab samples collected from normal individuals and those tested positive for SARS-CoV-2 by RTPCR, involving high throughput quantitative proteomics analysis. Several proteins like annexins, cytokines and histones were found differentially regulated in the host human cells following SARS-CoV-2 infection. Genes for these proteins were also observed to be differentially regulated when their expression was analyzed. Majority of the cytokine proteins were found to be up regulated in the infected individuals. Cell to Cell signaling interaction, Immune cell trafficking and inflammatory response pathways were found associated with the differentially regulated proteins based on network pathway analysis.

biochemistry↗

bioRxiv2021

Gramicidin S and Melittin - Potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection

The COVID19 pandemic has resulted in multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of gramicidin S and melittin peptides obtained from Bacillus brevis and bee venom respectively. Our in vitro antiviral assay results showed significant decrease in the viral load compared to the untreated group with no/very less cytotoxicity. The EC50 values for gramicidin S and melittin are calculated as 1.571g and 0.656g respectively. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 hours onwards with a maximal clearance after 24 hours post infection. Based on proteome analysis it was found that more than 250 proteins were found to be differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 hours post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.

cell biology↗

bioRxiv2021

Molecular Dynamics Simulations of hydrophobic peptides that form β-hairpin structures in solution

Peptides designed with residues that have high propensity to occur in {beta}-turns, form {beta}-hairpin structures in apolar solvents as well in polar organic solvents such as dimethyl sulfoxide (DMSO), methanol and varying percentages of DMSO in chloroform (CHCl3). Presumably due to limited solubility, their conformations have not been investigated by experimental methods in water. We have examined the conformations of such designed peptides that fold into well-defined {beta}-hairpin structures facilitated by {beta}-turns, in the crystalline state and in solution, by Molecular Dynamics Simulations (MDS). The peptides fold into {beta}-hairpin structures in water, starting from extended conformation. In DMSO, folding into {beta}-hairpin structures was not observed, starting from extended conformation. However, when the starting structure is in {beta}-hairpin conformation, unfolding is not observed during MDS in DMSO. Water clearly favours folding of short, hydrophobic peptides into {beta}-turn and {beta}-hairpin conformations from extended structures. DMSO does not have a denaturing effect on short, hydrophobic peptides.

biochemistry↗

bioRxiv2019

Glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis

The mouse embryo undergoes compaction at the 8-cell stage and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. We report here, that this first cell fate specification event is controlled by glucose metabolism. Glucose does not fuel mitochondrial ATP (energy) generation and glycolysis is dispensable for blastocyst formation. Glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1, and the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of AP-2{gamma}. YAP1, TEAD4 and AP-2{gamma} physically interact to form a nuclear complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, but this cascade of events constituting \"Developmental Metabolism\" specifically controls the fate of TE cells.

developmental biology↗