Biology subjects

Malik, N.

Publications and source records attributed to Malik, N..

14 recordsLinked to original sources

bioRxiv2025

DRP1-mediated mitochondrial dynamics orchestrate EMT in glioblastoma cells

BackgroundEpithelial to mesenchymal transition (EMT), a differentiation process, frequently imparts invasive properties in Glioblastoma Multiforme (GBM), which leads to a poor prognosis. Cells lose apical-basal polarity, cell-cell connections, and/or chemo-resistance during EMT, which can result in the spread of cancer and the acquisition of additional stem cell-like traits. It is unclear how organelle dynamics influence EMT in this respect. The interaction between cytoskeletal and mitochondrial regulators governing GBM cell EMT is explored in this article. Results and DiscussionIn GBM cells, we observed that TGF-{beta}-induced EMT led to a proliferative arrest, which was accompanied by a fragmented mitochondrial morphology, elevated expression of fission markers such as DRP1, MFF, and FIS1, and most importantly, localization of mitochondria near the cell boundaries. An increase in mitochondrial ROS accompanied this, but their functional status was indicated by a higher oxygen consumption rate (OCR). Additionally, cytoskeleton re-distribution and EMT reversal were the outcomes of si-RNA-mediated elimination of the fission-marker DRP-1 or pharmacological inhibition of fission by Mdivi-1. On the other hand, drugs that disrupt the cytoskeleton, shifted the spatial distribution of mitochondria to the perinuclear area, which had an adverse effect on EMT. Notably, it was shown that RhoA, a protein that helps organize the actin cytoskeleton, co-immunoprecipitates with DRP1 and governs both cytoskeletal dynamics and mitochondrial fission in its presence. ConclusionOur research sheds substantial insight on the current interactions between the cytoskeleton and mitochondrial spatial dynamics that control EMT in GBM cells, which may have significant therapeutic implications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/694080v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@81b796org.highwire.dtl.DTLVardef@3bec9aorg.highwire.dtl.DTLVardef@2a79c3org.highwire.dtl.DTLVardef@9ff14a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Differential expression of GABARAPs in GBM renders temozolomide sensitivity in a p53-dependent manner

Glioblastoma (GBM) is one of the most debilitating and extremely aggressive tumors, with a median survival of less than a year. GBMs have high metastatic potential and frequently acquire chemoresistance. The current multimodal treatment approaches for GBM include surgical tumor resurrection, radiotherapy, and chemotherapy but these approaches leave the patient with long-term disabilities such as depletion of cognitive abilities, leukoencephalopathy, and recurrence in 6-8 months. Glioma cells are highly dependent on autophagy to survive and proliferate. Autophagy inhibition has proven to be a beneficial strategy for restricting glioma growth. However, the autophagy pathway cannot be efficiently targeted due to the lack of specific autophagy inhibitors. Understanding the vulnerabilities in autophagy gene expression can help to design better autophagy inhibitors. This study demonstrates the differential expression of GABARAP family members in low-grade glioma and GBM. Our study highlights the differential expression of GABARAP family members in response to autophagy inhibition and induction. Moreover, the knockdown of specific GABARAP family members enhanced proliferation and reduced temozolomide (TMZ) sensitivity of glial cells by decreasing the p53 expression. The selective expression pattern of GABARAP genes in Glioblastoma can be utilized to screen for patients who might respond better to temozolomide treatment. The differential expression of GABARAP family members highlights the subtle regulation of the autophagy pathway in response to environmental cues.

cancer biology↗

bioRxiv2023

Post-mortem AT-8 reactive tau species correlate with non-plaque Aβ levels in the frontal cortex of non-AD and AD brains

The amyloid cascade hypothesis states that A{beta} and its aggregates induce pathological changes in tau, leading to formation of neurofibrillary tangles (NFTs) and cell death. A caveat with this hypothesis is the temporo-spatial divide between plaques and NFTs. This has been addressed by the inclusion of soluble species of A{beta} and tau in the revised amyloid cascade hypothesis, however, the demonstration of a correlative relationship between A{beta} and tau burden in post-mortem human tissue has remained elusive. Employing frozen and fixed frontal cortex grey and associated white matter tissue from non-AD controls (Con; n=39) and Alzheimers diseases (AD) cases (n=21), biochemical and immunohistochemical measures of A{beta} and AT-8 phosphorylated tau were assessed. Native-state dot-blot from crude tissue lysates demonstrated robust correlations between intraregional A{beta} and AT-8 tau, such increases in A{beta} immunoreactivity conferred increases in AT-8 immunoreactivity, both when considered across the entire cohort as well as separately in Con and AD cases. In contrast, no such association between A{beta} plaques and AT-8 were reported when using immunohistochemical measurements. However, when using the non-amyloid precursor protein cross reactive MOAB-2, antibody to measure intracellular A{beta} within a subset of cases, a similar correlative relationship with AT-8 tau as that observed in biochemical analysis was observed. Collectively our data suggests that accumulating intracellular A{beta} may influence AT-8 pathology. Despite the markedly lower levels of phospho-tau in non-AD controls correlative relationships between AT-8 phospho-tau and A{beta} as measured in both biochemical and immunohistochemical assays were more robust in non-AD controls, suggesting a physiological association of A{beta} production and tau phosphorylation, at least within the frontal cortex. Such interactions between regional A{beta} load and phospho-tau load may become modified with disease potentially, as a consequence of interregional tau seed propagation, and thus may diminish the linear relationship observed between A{beta} and phospho-tau in non-AD controls. This study provides evidence supportive of the revised amyloid cascade hypothesis, and demonstrates an associative relationship between AT-8 tau pathology and intracellular A{beta} but not extracellular A{beta} plaques.

neuroscience↗

bioRxiv2023

The atypical cadherin FAT1 is a novel regulator of STAT1, driving its pro-tumorigenic effect via the STAT1/PDCD4 axis in glioblastoma

FAT1 is an atypical cadherin that has been shown to act both as an oncogene and a tumour suppressor gene (TSG) in different tumor types. We have earlier shown that upregulated FAT1 acts as an oncogene in glial tumors by promoting pro-tumorigenic inflammation and EMT in primary human glioblastoma and in cell lines. One effect was through the suppression of the Tumor Suppressor Gene (TSG), Programmed Cell Death 4 (PDCD4). Here, we have studied how, in glioblastoma, upregulated FAT1 affects downstream events that control PDCD4 expression. In silico analysis of the PDCD4 promoter revealed multiple STAT1 binding sites. We also found a positive correlation in mRNA levels of STAT1 and FAT1 in the Glioblastoma databases, as well as in resected patient derived tumor samples by qPCR. Increased FAT1 as well as STAT1 were associated with poor prognosis in these data bases. In the glioblastoma cell lines LN229 and U87MG, FAT1 knockdown resulted in decreased STAT1 expression. Also, STAT1 knockdown resulted in increased PDCD4 expression, implying that STAT1 may mediate FAT1s role in suppressing PDCD4. Further, ChIP experiments showed that STAT1 protein binds to the PDCD4 promoter and upon FAT1 knockdown, STAT1 binding to the PDCD4 promoter reduces. As for FAT1, STAT1 knockdown also reduces the expression of pro-inflammatory cytokines and EMT markers, also migration and invasion of glioma derived cell lines. This work identifies STAT1 as a novel downstream mediator of FAT1 which mediates its pro-tumorigenic action in suppressing the TSG, PDCD4.

cancer biology↗

bioRxiv2023

In silico Antibody-Peptide Epitope prediction for Personalized cancer therapy

The human leukocyte antigen (HLA) system is a complex of genes on chromosome 6 in humans that encodes cell-surface proteins responsible for regulating the immune system. Viral peptides presented to cancer cell surfaces by the HLA trigger the immune system to kill the cells, creating Antibody-peptide epitopes (APE). This study proposes an in-silico approach to identify patient-specific APEs by applying complex networks diagnostics on a novel multiplex data structure as input for a deep learning model. The proposed analytical model identifies patient and tumor-specific APEs with as few as 20 labeled data points. Additionally, the proposed data structure employs complex network theory and other statistical approaches that can better explain and reduce the black box effect of deep learning. The proposed approach achieves an F1-score of 80% and 93% on patients one and two respectively and above 90% on tumor-specific tasks. Additionally, it minimizes the required training time and the number of parameters.

cancer biology↗

bioRxiv2022

An In Vivo Analysis of the Functional Motifs of DEAD-box RNA Helicase Me31B in Drosophila Fertility and Germline Development

In Drosophila germline, Me31B is a putative ATP-dependent, RNA helicase that plays role in post-transcriptional RNA regulation to ensure the correct spatial and temporal expression of the mRNAs, a process crucial for proper germline development and fertility. However, Me31Bs in vivo working mechanism remains unclear. In this study, we aim to analyze the functions of Me31Bs key domains/motifs to understand how these domains/motifs operate to fulfill the proteins overall activities. We generated Drosophila strains mutant for six important motifs including three ATPase/helicase motifs (DEAD-box, DVLARAK, and HRIGR), the N-terminal domain (N-ter), the C-terminal domain (C-ter), and a protein-binding motif (FDF motif-binding motif). In characterizing these mutants, we observed that the three ATPase/helicase motif mutations cause dominant female sterility which is associated with developmental defects in oogenesis and embryogenesis. Follow-up examination of the DVLARAK motif mutant revealed its abnormalities in germline mRNA localization and transcript level. The Me31B N-ter domain (deletion of C-ter), C-ter domain (deletion of N-ter), and mutation of FDF motif-binding motif led to a decrease in female fertility and abnormal subcellular Me31B localizations in the egg chambers. Moreover, deletion of Me31B N-ter or C-ter motif results in a decrease of Me31B protein levels in the ovaries. This study indicates that these six motifs of Me31B play different roles to contribute to Me31Bs whole-protein functions like ATPase, RNA helicase, protein stability, protein localization, and partner protein binding, which are crucial for germline development and fertility. Considering Me31B protein familys conserved presence in both Drosophila germline and soma (for example, neurons) and in other organisms such as yeast, worm, mouse, and human, the results from this study could expand our understanding of Me31B helicase familys general working mechanisms in different cell types and species.

developmental biology↗

bioRxiv2022

PKCβ facilitates leukemogenesis in chronic lymphocytic leukaemia by promoting constitutive BCR-mediated signaling

B cell antigen receptor (BCR) signaling competence is critical for pathogenesis of chronic lymphocytic leukemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKC function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKC{beta}II upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKC-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKC-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKC-KR expressing cells, coincident with upregulation of PKC{beta}II: supported by activation of key signaling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKC{beta}II inhibitor) treatment of PKC-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKC{beta}II in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKC-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKC{beta} expression facilitates leukemogenesis and identify that BCR-mediated signaling is a key driver of CLL development in the PKC-KR model. Statement of SignificancePKC{beta} facilitates leukemogenesis of CLL, driven through an SP1-regulated transcriptional program and promotes BCR signaling. Thus far, PKC{beta} is the only kinase within the BCR signaling pathway, a key pathway in driving CLL pathogenesis, implicated in the generation of neoplastic B lineage cells.

cancer biology↗

bioRxiv2022

Intrinsic disorder in CENP-ACse4 tail and its chaperone facilitates synergistic association for kinetochore stabilization

The kinetochore is a complex multiprotein network that assembles at a specialized DNA locus called the centromere to ensure faithful chromosome segregation. The centromere is epigenetically marked by a histone H3 variant - the CenH3. The budding yeast CenH3, called Cse4, consists of an unusually long and disordered N-terminal tail that has a role in kinetochore assembly. Its disordered chaperone, Scm3 is involved in its centromeric deposition as well as in the maintenance of a segregation-competent kinetochore. The dynamics of the Cse4 N-tail and chaperone interaction have not been studied, leaving a gap in our understanding of their roles at the centromere. Previously, we had shown that Scm3 is an intrinsically disordered protein. Here, using NMR and a variety of biophysical and bioinformatics tools, we show that Cse4 N-tail is also disordered, the two proteins interact with each other at multiple sites, and this interaction reduces the disorder in Scm3; the chain opens up relative to the native state ensemble and the conformational exchange is reduced. Interestingly, this interaction between the two intrinsically disordered protein is fairly specific as seen by positive and negative controls, and is majorly driven by electrostatics as both the proteins have multiple acidic and basic regions. The complex retains a fair amount of disorder, which facilitates a synergistic association with the essential inner kinetochore Ctf19-Mcm21-Okp1-Ame1 complex; a model has been suggested to this effect. Given the abundance of intrinsic disorder in the kinetochore proteins, this type of interaction and adaptation may be prevalent in other proteins as well for mediating kinetochore assembly. Thus, the present study, on one hand, provides significant structural and mechanistic insights into the complex and dynamic process of kinetochore assembly, and on the other hand, illustrates a mechanism that intrinsically disordered proteins would adapt to mediate the formation of complex multiprotein networks, in general. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504061v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8fe5cforg.highwire.dtl.DTLVardef@138afb1org.highwire.dtl.DTLVardef@96943forg.highwire.dtl.DTLVardef@1deb673_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

mTORC1 activity is essential for disease progression in chronic lymphocytic leukemia

The precise role of mechanistic target of rapamycin complex 1 (mTORC1) during chronic lymphocytic leukemia (CLL) pathogenesis remains to be elucidated. Targeted deletion of mTORC1 component Raptor in adult mice reveals that mTORC1 function is essential for initiation and maintenance of CLL. Raptor-deficient bone marrow-derived PKC-KR transduced haemopoietic progenitors failed to generate a CLL-like disease in vitro, due to an inability to overcome the mTORC1-mediated block in B cell lineage commitment. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor BM-derived PKC-KR transduced cells after disease was established, revealed a reduced CLL-like disease load and a significant increase in survival in the mice. Interestingly in mice transplanted with an aggressive CLL-like disease, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive leukemic disease. Rapamycin efficiently targeted the translation elongation axis eEF2/eEF2K downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. Rapamycin treatment of primary CLL cells halted proliferation, modulated eEF2K/eEF2 phosphorylation and inhibited MCL1 expression. Our studies demonstrate that mTORC1 plays an essential role in leukemia progression in vitro and in vivo in our CLL mouse model, with evidence for increased rapamycin sensitivity in aggressive secondary CLL transplants. Furthermore, the suppression of translation elongation through inactivation of eEF2 may offer a novel therapeutic target for blocking CLL progression.

cancer biology↗

bioRxiv2022

GWAShub: A Web-based Resource to Democratize Genome-Wide Association Studies in Crop Plants

Genome-wide association study (GWAS) is a popular approach for linking natural genetic variation with phenotype variation and thus is central to crop quantitative genetics. The ever-increasing wealth of publicly available genomic sequence information for crop plants presents an unprecedented opportunity for utilizing GWAS for the identification of genes governing a plethora of agronomic traits. However, the lack of technical expertise and computational infrastructure is still hindering the ability of plant breeders to conduct GWAS in a self-reliant manner. Here, we present a GWAShub web server that provides a user-friendly interface for performing comprehensive GWAS and post-GWAS in crop plants utilizing publicly available genomic sequence variation data, comprehensive annotation data and diverse computational tools. The utility of GWAS-hub was further demonstrated by conducting large-scale GWAS for flowering/maturity time traits in chickpea. This analysis identified three different flowering/maturity time associated genes, all encoding different histone methyltransferases. Thus, epigenetic regulation is identified as vital mechanism regulating flowering time and maturity duration in chickpea. Finally, we hope GWAShub (www.gwashub.com) will enable resource-scarce researchers to join the GWAS revolution fueled by advancements in next-generation sequencing and computational genomics

bioinformatics↗

bioRxiv2020

Strategies for identifying dynamic regions in protein complexes: flexibility changes accompany methylation in chemotaxis receptor signaling states

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylation mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate dynamics. The strategies used in the study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/974428v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8a8b0forg.highwire.dtl.DTLVardef@1afb646org.highwire.dtl.DTLVardef@e7dbaaorg.highwire.dtl.DTLVardef@685c0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReceptors exhibit greater ns timescale dynamics in unmethylated vs methylated state C_LIO_LIMethylation helix 2 likely involved in increased flexibility of unmethylated state C_LIO_LIDynamics occur on multiple timescales in both states of the receptor C_LI

biochemistry↗

bioRxiv2019

Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is upregulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including 4 endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are poorly phosphorylated by Akt as they possess an n+1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

biochemistry↗

bioRxiv2019

Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukaemia cells

p53 is a tumor suppressor, which belongs to the p53 family of proteins. The family consists of p53, p63 and p73 proteins, which share similar structure and function. Activation of wild-type p53 or TAp73 in tumors leads to tumor regression, and small molecules restoring the p53 pathway are in clinical development. Protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid, is a clinically approved drug applied in photodynamic diagnosis and therapy. PpIX induces p53- and TAp73-dependent apoptosis and inhibits TAp73/MDM2 and TAp73/MDM4 interactions. Here we demonstrate that PpIX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and activates apoptosis in B-cell chronic lymphocytic leukaemia cells without illumination and without affecting normal cells. PpIX stabilizes p53 and TAp73 proteins, induces p53-downstream apoptotic targets and provokes cancer cell death at doses non-toxic to normal cells. Our findings open up new opportunities for repurposing PpIX for treating lymphoblastic leukaemias with wtTP53.

cancer biology↗

bioRxiv2018

Structural plasticity of CENP-A regulated by H4 influences cellular levels and kinetochore assembly

The Histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast. The level of Cse4 in cells is tightly regulated, primarily by ubiquitin-mediated proteolysis. However, the structural transitions in Cse4 that regulate centromere localization and interaction with regulatory components are poorly understood. Using time resolved fluorescence, NMR and molecular dynamics we show for the first time that soluble Cse4 can exist in a closed conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner H4, alters the inter-domain interaction within Cse4, ensuring an open state that will lend itself to proteolysis. This dynamic model allows kinetochore formation only in presence of H4, as the N-terminus, which is required for interaction with centromeric components will be unavailable in absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a unique structure-based regulatory mechanism for Cse4 localization and prevention of premature kinetochore assembly.

biophysics↗