Biology subjects

Shukla, S.

Publications and source records attributed to Shukla, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

PKM2-mediated epigenetic reprogramming regulates hypoxic expression of PFKFB3 to promote breast cancer progression

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms existing between the key glycolytic enzymes in hypoxic cancer cells are largely unexplored. In particular, M2 isoform of pyruvate kinase (PKM2) - the ratelimiting enzyme of glycolysis, is well-known to confer adaptive advantages under hypoxia. Herein, we report a non-canonical role of PKM2 in functioning as a co-activator of HIF-1 to govern the transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Nuclear PKM2 enhances HIF-1 and p300 occupancy at PFKFB3 hypoxia responsive elements (HREs) resulting in its upregulation. Consequently, absence of nuclear PKM2 fails to recruit HIF-1 which activates an opportunistic occupancy of HIF-2 at PFKFB3 HREs. Enhanced binding of HDAC3 also occurs in the absence of PKM2 which prevents HIF-2 from efficiently inducing PFKFB3 to hamper proliferation of hypoxic breast cancer cells. In addition, clinical relevance of the study has been investigated by demonstrating that Shikonin blocks nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, MCF7 cells-derived xenograft tumors in mice exhibited substantial tumor growth inhibition when treated with shikonin, highlighting the vitality of targeting PKM2. Taken in concert, this work provides novel insights into contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported molecular axis exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression essential for achieving high glycolytic flux. Statement of significanceNuclear PKM2 orchestrates the binding of histone modifiers to epigenetically alter PFKFB3 promoter and affects the binding of HIF-2. Notably, targeting this axis attenuates proliferation of hypoxic breast cancer cells.

cancer biology↗

bioRxiv2022

Development of Machine Learning Model for Pan-cancer Subgroup Identification using Multi-omics Data

AO_SCPLOWBSTRACTC_SCPLOWCancer is a heterogeneous disease and patients with tumors from different organs can share similar epigenetic and genetic alterations. Therefore, it is crucial to identify the novel subgroup of patients with similar molecular characteristics. It is possible to propose a better treatment strategy when the heterogeneity of the patient is accounted for during subgroup identification irrespective of the tissue of origin. In this work, mRNA, miRNA, DNA methylation, and protein expression features from pan-cancer samples were concatenated and non-linearly projected to lower dimension using machine learning (ML) algorithm. This data was then clustered to identify multi-omics based novel subgroups. The clinical characterization of these ML subgroups indicated significant differences in overall survival (OS) and disease free survival (DFS) (p-value<0.0001). The subgroups formed by the patients from different tumors shared similar molecular alterations in terms of immune microenvironment, mutation profile, and enriched pathways. Further, decision-level and feature-level fused classification models were built to identify the novel subgroups for unseen samples. Additionally, the classification models were used to obtain the class labels for the validation samples and the molecular characteristics were verified. To summarize, this work identified novel ML subgroups using multi-omics data and showed that patients with different tumor types can be similar molecularly. We also proposed and validated the classification models for subgroup identification. The proposed classification models can be used to identify the novel multi-omics subgroups and the molecular characteristics of each subgroup can be used to design appropriate treatment regimen.

bioinformatics↗

bioRxiv2022

Data-Driven Design of Protein-Derived Peptide Multiplexes for Biomimetic Detection of Exhaled Breath VOC Profiles

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect the characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposures assessment.

bioengineering↗

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

iOBPdb: A Database for Experimentally Determined Functional Characterization of Odorant Binding Proteins

Summary/AbstractOdorant binding proteins, OBPs, are a diverse family of small, globular, extra-cellular proteins solubilize volatile organic compounds (VOCs) so they can be internalized and transported by an organism. Since their initial discovery in the early eighties 1, thousands of OBPs have been identified through genome sequencing and characterized by fluorescence ligand binding assays 2. While individual OBPs have been studied in the context of their roles in specific organism, there have been no studies towards the understanding of the comparative structure-function relations of all known OBPs, primarily due to a lack of a centralized database that incorporates the binding affinity with the structure of all OBPs. Incorporating OBP information into a database requires not only an extensive search of all existing resources, but also creating a useful platform that relates sequence structures to target functions. Combining 215 functional studies containing 381 unique OBPs from 91 insect species we created a database, iOBPdb: https://iobpdb.herokuapp.com, of OBP binding affinities for a wide range of VOC targets. We demonstrate here that the construction of this initial database provides powerful search and associative capabilities including interrogating odor binding proteins as clusters and groups by sequence similarity versus protein and target molecular weights, and by the functional groups of the VOC targets. The comparative interrogation of the probe-target recognition allows for a more comprehensive understanding of the underlying structural features of all OBPs that had not been possible by only examining the OBPs individually. We present our results in a variety of phylogenetic representations as well as providing the binding profiles of OBP groups to VOC functional moieties. Potential applications include development of molecular probes for biosensors, novel bioassays and drugs, discovery of novel pesticides which inhibit VOC / OBP interactions, as well providing a foundational basis for the functional understanding of odor sensing and perception in the brain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/498339v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@9c2fa0org.highwire.dtl.DTLVardef@1fad38aorg.highwire.dtl.DTLVardef@56175forg.highwire.dtl.DTLVardef@189797_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Machine learning approaches for classificationof Plasmodium falciparum life cycle stagesusing single-cell transcriptomes

Malaria, spread by the female Anopheles mosquito, is a highly fatal disease widespread in many parts of the world, causing 0.4 million deaths globally. Vital gene expressions form the basis in the detection of malaria infection levels. Quantification of malaria parasite infected RBCs and classification of its life cycle stages are done at macroscopic level by experts, for making informed decisions. Off late multiple computational approaches have been proposed to circumvent the problem of dimensionality leading to accurate predicted results. In this work a dimensionality reduction technique based on Genetic Algorithm (GA) is applied on P. falciparum single-cell transcriptomics to arrive at an optimized subset of features from the larger dataset. Features are chosen based on their class variants considering increased efficiency and accuracy, to separately transform the selected elements into a lower dimension. For the classification of the life cycle of malaria parasite based on single cell transcriptome data, a three-pronged approach employing the multiclass Support Vector Machine (SVM), Logistic Regression (LR) and Random Forest (RF) techniques is used. Distribution of cells was visualised and mapped using the R-based Seurat package. Further, we constructed protein interaction networks of the genes identified by the feature selection method and elucidated the role of the proteins in progression of the parasite through its life cycle. Our approach presents a novel protocol to implement ML techniques on scRNA seq datasets and subsequently harnessing the extracted information for biomarker/drug target detection.

bioinformatics↗

bioRxiv2022

RNA-protein interactome at the Hepatitis E virus internal ribosome entry site

Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to utilize the host translation machinery. Earlier studies have shown that genotype 1-Hepatitis E virus (g1-HEV) utilizes both cap-dependent and cap-independent translation machineries for its replication and proliferation. Cap-independent translation in g1-HEV is driven by an eighty seven nucleotide-long RNA element which acts as a noncanonical, internal ribosome entry site like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study reveals indispensable roles of host ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating efficient translation from the IRESl element and establish the function of HEV IRESl as a bonafide internal ribosome entry site. Author summaryProtein synthesis is a fundamental process for survival and proliferation of all living organisms. Majority of cellular proteins are produced through cap-dependent translation. Cells also utilize a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus is a major cause of hepatitis worldwide. The viral genome is a capped positive strand RNA. Viral non-structural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth ORF in genotype 1-HEV, which produced the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data proves that HEV-IRESl is a bonafide internal ribosome entry site.

microbiology↗

bioRxiv2022

In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair

The endosomal sorting complex required for transport (ESCRT) machinery has been shown to be centrally involved in repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open a new avenue for mechanistic understanding of endolysosomal membrane repair. Significance statementOne of the ways by which protein aggregates can propagate and lead to progression of a neurodegenerative disease is by damaging the membrane that is destined to degrade the misfolded, aggregated protein. ESCRT machinery has been implicated in sealing these damaged membranes, and the nature of the membrane recruitment trigger signal for this machinery is a major open question. Here, we show in vitro that ALG-2 can bring ESCRT machinery to membranes in a Ca2+-dependent manner.

cell biology↗

bioRxiv2021

B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/{Delta}157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (Covishield)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/{Delta}157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/{Delta}157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/{Delta}157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/{Delta}157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

microbiology↗

bioRxiv2021

Particle Uptake Driven Phagocytosis in Macrophages and Neutrophils Enhances Bacterial Clearance

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

immunology↗

bioRxiv2021

TLR2 on CD4+ and CD8+ T cells promotes late control of Mycobacterium tuberculosis infection

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout (ko) mice is due to its role in macrophages, on T cells or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro-(IFN-{gamma}, TNF-) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells did not affect early control but did result in decreased late control of Mtb in the lungs of infected mice. This suggests that T cell co-stimulation by mycobacterial TLR2 ligands in vivo is important for control of infection during the chronic phase of Mtb infection in the lung.

immunology↗

bioRxiv2021

IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs provide an opportunity to inform fundamental hypotheses in developmental programs and help accelerate the design of stem cell-based technologies. We first describe the architecture of IQCELL. Next, we apply IQCELL to a scRNA-seq dataset of early mouse T-cell development and show that it can infer a priori over 75% of causal gene interactions previously reported via decades of research. We will also show that dynamic simulations of the derived GRN qualitatively recapitulate the effects of the known gene perturbations on the T-cell developmental trajectory. IQCELL is applicable to many developmental systems and offers a versatile tool to infer, simulate, and study GRNs in biological systems. (https://gitlab.com/stemcellbioengineering/iqcell)

systems biology↗

bioRxiv2021

Individual Molecular Motors use Low Forces to bypass Roadblocks during Collective Cargo Transport

A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

biophysics↗

bioRxiv2020

In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and hnRNP-1

Novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 1.5 million lives worldwide and counting. As per the GISAID database, the genomics of SARS-CoV2 is extensively studied with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, researchers have focused a lot on missense variants but the least work is done on the UTRs. One of the most frequent 5 UTR variants in the SARS-CoV-2 genome is the C241T with a global frequency of more than 0.9. In the present study, the effect of the C241T mutation has been studied with respect to change in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem loops with host transcription factor MADP1 indicating reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.

bioinformatics↗

bioRxiv2020

Unique mutational changes in SARS-CoV2 genome of different state of India

COVID-19 is a global pandemic causing more than 8 million deaths till mid-August, 2020. In India, more than 3 million confirmed cases have been reported although with relatively low death rate of 1.8%. In this study, we sequenced 47 genomes of SARS-CoV-2 from the patients of 13 districts of Uttar Pradesh (UP), the largest state of India using third-generation sequencing technique. The phylogenetic clustering revealed that no UP sample was aligned with the previously defined USA clade, where the mortality was high. We identified 56 distinct SNP variations in the genomes of UP resulting in a unique mutation rate of 1.19% per sequence, which is greater than the value 0.88% obtained for the rest of India. The relatively less death rate in UP indicates that the mutation in the virus is deleterious. Further investigation is required with larger sample size to determine the degree of virulence vis-a-vis SNP variation.

bioinformatics↗

bioRxiv2020

Studies on C. elegans probed with estradiol, elucidate the critical role of Na+/H+ exchanger, nhx-2 in reproductive senescence and neuronal health

Setting in of reproductive senescence (RS) gives rise to several changes, making aged individuals susceptible to multiple disorders including neurodegenerative diseases, cardiovascular ailments and bone disorders amongst others. The present study, employing transgenic C. elegans that expresses human alpha synuclein, endeavors to decipher the association of reproductive senescence with age-associated neurodegenerative diseases and behavioral ageing, under normal conditions and after being probed with estradiol. We carried out RNAi induced silencing of a subset of 22 genes that are known to delay RS, followed by studies on alpha-Synuclein aggregation and associated effects. These studies led us to functional characterization of the Na+/H+ exchanger; nhx-2, expressed exclusively in gut. We found that RNAi of nhx-2 not only ameliorates the effects associated with alpha-Synuclein aggregation, but it also attunes effects related to behavioral aging including that of reproductive health-span and neuroprotection via mimicking dietary restriction, as it alters food absorption from the gut. We further elucidated that these effects are Sir-2.1 driven as nhx-2 knock out did not delay reproductive senescence in knock down condition of sir-2.1. To substantiate our findings, we performed whole transcriptome analysis in nhx-2 mutant strain. Our data revealed differential expression of 61 out of 62 hallmark genes of CR described by GenDR, in knock out condition of nhx-2. As estradiol plays a central role in both reproductive health as well as neuronal health, we subjected worms to exogenous estradiol treatment and observed that it led to elevated levels of nhx-2. Studies on structural binding analysis demonstrated significant binding potential of estradiol receptor NHR-14 with nhx-2 gene and ChIP analysis revealed that estradiol treatment gives rise to enhanced NHX-2 levels through inducing the promoter specific histone H3 acetylation (H3K9) and lysine methylation (H3K4me3). These studies identify nhx-2 as an important modulator that extends reproductive longevity and ameliorates effects associated with alpha synuclein aggregation in C elegans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/229344v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@917377org.highwire.dtl.DTLVardef@133a07dorg.highwire.dtl.DTLVardef@6d6f28org.highwire.dtl.DTLVardef@9cde9a_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISilencing of sodium proton antiporter nhx-2 affords neuroprotection and ameliorates effects associated with alpha-Synuclein aggregation via mimicking dietary restriction in C. elegans. C_LIO_LIExogenous 17-{beta}-Estradiol treatment induces the expression of nhx-2, through inducing the promoter specific histone H3 acetylation (H3K9) and lysine methylation (H3K4me3). C_LIO_LIEffects associated with nhx-2, including prolonged reproductive span and neuroprotective effects, are SIR-2.1 driven. C_LIO_LInhx-2 silencing decreases alpha-Synuclein aggregation, however; estradiol mediated overexpression above the endogenous levels, does not amend the aggregation any further. C_LI

neuroscience↗

bioRxiv2020

Comprehensive and Integrated Genomic Characterization of Human Immunome in Cancer

Genetic alterations in immune-related pathways are common hallmarks of cancer. However, to realize the full potential of immunotherapy, a comprehensive understanding of immune networks and how mutations impact network structure and functional output across cancer types is instrumental. Herein we systematically interrogated somatic mutations that could express neoantigens and alter immune responses in cancer patients compared to wild-type controls. To do so, we developed a network-based immunogenomics model (NIPPER) with scoring systems to prioritize critical genes and mutations eliciting differential HLA binding affinity and alternate responses to immunotherapy. These mutations are enriched in essential protein domains and often alter tumor infiltration by immune cells, affecting T cell receptor repertoire and B cell clonal expansion. Furthermore, we devised an interactome network propagation framework integrated with drug associated gene signatures to identify potential immunomodulatory drug candidates. Together, our systems-level analysis results help interpret the heterogeneous immune responses among patients, and serve as a resource for future functional studies and targeted therapeutics. SignificanceCancer cells induce specific immune-related pathway perturbations by mutations, transcriptional dysregulation, and integration of multi-omics data can help identify critical molecular determinants for effective targeted therapeutics.

immunology↗

bioRxiv2020

Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

We previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

cancer biology↗