Biology subjects

Nayak, S.

Publications and source records attributed to Nayak, S..

45 records · Page 3Linked to original sources

bioRxiv2020

Identification of PIM1 substrates reveals a role for NDRG1 in prostate cancer cellular migration and invasion

PIM1 is an oncogenic serine/threonine kinase that promotes and maintains prostate tumorigenesis. To more fully understand the mechanism by which PIM1 promotes oncogenesis, we performed a chemical genetic screen to identify direct PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in the suppression of cancer cell invasion and metastasis. NDRG1 is phosphorylated by PIM1 at serine 330 (pS330), and the level of NDRG1 pS330 is associated with high grade compared to low grade prostate tumors. While NDRG1 pS330 is largely cytoplasmic, total NDRG1 is both cytoplasmic and nuclear. Mechanistically, PIM1 phosphorylation of NDRG1 decreases its stability, reducing its interaction with AR, and thereby lowering expression of AR target genes. PIM1-dependent NDRG1 phosphorylation also reduces NDRG1s ability to suppress prostate cancer cell migration and invasion. Our study identifies a novel set of PIM1 substrates in prostate cancer cells using a direct, unbiased chemical genetic screen. It also provides key insights into the mechanisms by which PIM1-mediated phosphorylation of NDRG1 impairs its function, resulting in enhanced cell migration and invasion.

cancer biology↗

bioRxiv2019

Replication gaps underlie BRCA-deficiency and therapy response

Cancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, \"BRCAness,\" and we propose are fundamental to the mechanism of action of genotoxic chemotherapies.

cancer biology↗

bioRxiv2019

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

cancer biology↗

bioRxiv2019

Replication gaps are a cancer vulnerability counteracted by translesion synthesis

The replication stress response which serves as an anti-cancer barrier is activated not only by DNA damage and replication obstacles, but also oncogenes, mystifying how cancer evolves. Here, we identify that oncogene expression, similar to cancer therapies, induces single stranded DNA (ssDNA) gaps that reduce cell fitness, unless suppressed by translesion synthesis (TLS). DNA fiber analysis and electron microscopy reveal that TLS restricts replication fork slowing, reversal, and fork degradation without inducing replication fork gaps. Evidence that TLS gap suppression is fundamental to cancer, a small molecule inhibitor targeting the TLS factor, REV1, not only disrupts DNA replication and cancer cell fitness, but also synergizes with gap-inducing therapies. This work illuminates that gap suppression during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

cancer biology↗

bioRxiv2019

Comparative evaluation of full-length isoform quantification from RNA-Seq

Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are short. Here we use simulated benchmarking data that reflects many properties of real data, including polymorphisms, intron signal and non-uniform coverage, allowing for systematic comparative analyses of isoform quantification accuracy and its impact on differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a simple approach is included as a baseline control. Salmon, kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform dramatically better than the simple approach. We determine the structural parameters with the greatest impact on quantification accuracy to be length and sequence compression complexity and not so much the number of isoforms. The effect of incomplete annotation on performance is also investigated. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification and isoform level DE should still be employed selectively.

bioinformatics↗

bioRxiv2019

Unique PKD like protein kinase in Entamoeba histolytica regulates vital actin mediated processes

Entamoeba histolytica causes widespread amoebiasis in humans. Multiple lines of emerging evidence have identified a repertoire of proteins involved in the process of erythrophagocytosis. However, the early initiation of the erythrophagosome at the site of erythrocytes attachment is not well understood. Here in our study we have identified and characterized a small Protein kinase D like protein (EhPKDL) in Eh, which nucleates actin polymerization and thus mediates many vital processes in Eh including erythrophagocytosis. Following multiple biochemical and biophysical approaches, we have characterized EhPKDL and have shown that EhPKDL can indeed interact and prime the nucleation of monomeric actin for polymerization. Furthermore, we went on to demonstrate the vitality of the EhPKDL in major actin-mediated processes like capping, motility, and erythrophagocytosis following knockdown of EhPKDL in the cellular context. Our study thus provides novel insights into the early actin nucleation in Eh and thus bridges the gap with our previous understanding of the assembly of erythrophagosomes.

cell biology↗

bioRxiv2018

Circadian control of lung inflammation in influenza infection

Influenza is a leading cause of respiratory mortality and morbidity. While inflammation is necessary for fighting infection, a fine balance of anti-viral defense and host tolerance is necessary for recovery. Circadian rhythms have been known to modulate inflammation. However, the importance of diurnal variability in the timing of influenza infection is not well understood. Here we demonstrate that endogenous rhythms influence the cellular response to infection in bronchoalveolar lavage (BAL), the pulmonary transcriptomic profile and lesional histology. This time dependent variability does not reflect alterations in viral replication. Rather, we found that better time-dependent outcomes were associated with a preponderance of NK and NKT cells and lower proportion of monocytes in the lung. Thus, host tolerance, rather than viral burden underlies the diurnal gating of influenza induced lung injury.\n\nSignificance statementOur work demonstrates the importance of circadian rhythms in influenza infection --a condition with significant public health implications. Our findings, which establish the role of the circadian rhythms in maintaining the balance between host tolerance pathways and anti-viral responses confers a new framework for evaluating the relevance of circadian influences on immunity.

immunology↗

bioRxiv2018

Iso-relevance Functions - A Systematic Approach to Ranking Genomic Features by Differential Effect Size

It is common to measure large numbers of features to identify those differing between experimental conditions; for example using RNA-Seq to search for differentially expressed genes. Ranking by p-value allows for statistical control, but has well known issues: unreliability without many replicates; and significance of biologically irrelevant effect sizes. As a result prioritization is typically performed in conjunction with effect size; the canonical one being "fold-change" [Formula]. However fold-change has several issues: division by zero, sensitivity to small values in the denominator, insensitivity to magnitude (1 over 2 equals 100 over 200). To mitigate these problems adding 1 to all values is a widely used heuristic; which we show using real and simulated data is typically highly sub-optimal, while the value 20 is nearly optimal in all cases. From another point of view, adding a fixed "pseudocount" to all values is essentially re-defining effect size from fold-change to something else. To explore this further, we axiomatize the concept of effect size and use this mathematical framework to study the problem in general. We also present the remarkable finding that pseudocounts strike a balance between sorting by fold-change and sorting by difference. Therefore, optimization is equivalent to finding the most harmonious balance between these two extremes. Lastly, the framework is illustrated on a fundamentally different type of problem, that of ranking di-codons by their differential abundance in the ORFeome of different species, where p-values are unavailable and one must solve the problem directly with effect sizes.

bioinformatics↗

bioRxiv2017

LINE-1 and the cell cycle: protein localization and functional dynamics

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology to aid its proliferation. However, a clear understanding of L1s lifecycle and the processes involved in restricting its insertion and its intragenomic spreading remains elusive. Here we identify modes of L1 proteins entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

cell biology↗