Biology subjects

Shukla, D.

Publications and source records attributed to Shukla, D..

79 records · Page 5Linked to original sources

bioRxiv2020

TLmutation: predicting the effects of mutations using transfer learning

A reoccurring challenge in bioinformatics is predicting the phenotypic consequence of amino acid variation in proteins. With the recent advancements in sequencing techniques, sufficient genomic data has become available to train models that predict the evolutionary statistical energies, but there is still inadequate experimental data to directly predict functional effects. One approach to overcome this data scarcity is to apply transfer learning and train more models with available datasets. In this study, we propose a set of transfer learning algorithms we call TLmutation, which implements a supervised transfer learning algorithm that transfers knowledge from survival data of a protein to a particular function of that protein. This is followed by an unsupervised transfer learning algorithm that extends the knowledge to a homologous protein. We explore the application of our algorithms in three cases. First, we test the supervised transfer on 17 previously published deep mutagenesis datasets to complete and refine missing datapoints. We further investigate these datasets to identify which mutations build better predictors of variant functions. In the second case, we apply the algorithm to predict higher-order mutations solely from single point mutagenesis data. Finally, we perform the unsupervised transfer learning algorithm to predict mutational effects of homologous proteins from experimental datasets. These algorithms are generalized to transfer knowledge between Markov random field models. We show the benefit of our transfer learning algorithms to utilize informative deep mutational data and provide new insights into protein variant functions. As these algorithms are generalized to transfer knowledge between Markov random field models, we expect these algorithms to be applicable to other disciplines.

bioinformatics↗

bioRxiv2019

Independence of HIF1a and androgen signaling pathways in prostate cancer

Androgen signaling drives prostate cancer progression and is a therapeutic target. Hypoxia/HIF1a signaling is associated with resistance to hormone therapy and a poor prognosis in patients treated with surgery or radiotherapy. It is not known whether the pathways operate in cooperation or independently. Using LNCaP cells with and without stable transfection of a HIF1a expression vector, we show that combined AR and HIF1a signaling promotes tumor growth in vitro and in vivo, and the capacity of HIF1a to promote tumor growth in the absence of endogenous androgen in vivo. Gene expression analysis identified 7 genes that were upregulated by both androgen and HIF1a. ChIP-Seq analysis showed that the AR and HIF/hypoxia signaling pathways function independently regulating the transcription of different genes with few shared targets. In clinical datasets elevated expression of 5 of the 7 genes was associated with a poor prognosis. Our findings suggest that simultaneous therapeutic inhibition of AR and HIF1a signaling pathways should be explored as a potential therapeutic strategy.

cancer biology↗

bioRxiv2019

Molecular Mechanism of Brassinosteroids Perception by the Plant Growth Receptor BRI1

Brassinosteroids (BRs) are essential phytohormones which bind to the plant receptor, BRI1, to regulate various physiological processes. The molecular mechanism of the perception of BRs by the ectodomain of BRI1 remains not fully understood. It also remains elusive why a substantial difference in biological activity exists between the BRs. In this work, we study the binding mechanisms of the two most bioactive BRs, brassinolide (BLD) and castasterone (CAT) using molecular dynamics simulations. We report free energy landscapes of the binding processes of both ligands as well as detailed ligand binding pathways. Our results suggest that CAT has lower binding affinity compared to BLD due to its inability to form hydrogen bonding interactions with a tyrosine residue in the island domain of BRI1. We uncover a conserved non-productive binding state for both BLD and CAT, which is more stable for CAT and may further contribute to the bioactivity difference. Finally, we validate past observations about the conformational restructuring and ordering of the island domain upon BLD binding. Overall, this study provides new insights into the fundamental mechanism of the perception of two most bioactive BRs, which may create new avenues for genetic and agrochemical control of their signaling cascade.

plant biology↗

bioRxiv2019

Molecular Basis of the Activation and Dissociation of Dimeric PYL2 Receptor in Abscisic Acid Signaling

Phytohormone abscisic acid (ABA) is essential for plant responses to biotic and abiotic stresses. Dimeric receptors are a class of ABA receptors that are important for various ABA responses. While extensive experimental and computational studies have investigated these receptors, it remains not fully understood how ABA leads to their activation and dissociation for interaction with downstream phosphatase. Here, we study the activation and the homodimeric association processes of PYL2 receptor as well as its heterodimeric association with the phosphatase HAB1 using molecular dynamics simulations. Free energy landscapes from ~223 s simulations show that dimerization substantially constrains PYL2 conformational plasticity and stabilizes inactive state, resulting in lower ABA affinity. Also, we establish the thermodynamic model for competitive binding between homodimeric PYL2 association and heterodimeric PYL2-HAB1 association in the absence and presence of ABA. Our results suggest that the binding of ABA destabilizes PYL2 complex and further stabilizes PYL2-HAB1 association, thereby promoting PYL2 dissociation. Overall, this study explains several key aspects on activation of dimeric ABA receptors, which provide new avenues for selective regulation of these receptors.

plant biology↗

bioRxiv2019

How do brassinosteroids activate their receptors?

Brassinosteroids (BRs) are an important class of plant growth hormones which signal through BRI1 and BAK1, leucine-rich repeat receptor-like kinases (LRR-RLKs). When bound to the BRI1 island domain, BRs act as a \"molecular glue\", mediating interactions between BRI1 and BAK1 extracellular domains. However, it is unclear how much other factors contribute to BR-induced BRI1-BAK1 association, including stabilization of the BRI1 island domain and large conformational changes in BRI1. We use several molecular dynamics simulation-based methods to explore the contributions of each mechanism to BR-dependent Arabidopsis thaliana BRI1-BAK1 association. We find that specific BR interactions make major contributions to BRI1-BAK1 association free energy. BR binding stabilizes the BRI1 island domain, while BRI1 undergoes a large conformational change to form a secondary interface with BAK1. These results suggest that each mechanism plays a part in BR signal transduction while raising questions about the functional role of conformational dynamics in other LRR-RLKs.

plant biology↗

bioRxiv2018

Concestor kinase activation mechanism uncovers the cyclin dependence of CDK family kinases.

Evolution has altered the free energy landscapes of protein kinases to introduce different regulatory switches and alters their catalytic functions. An understanding of evolutionary pathways behind these changes at atomistic resolution is of great importance for drug design. In this work, we demonstrate how cyclin dependency has emerged in cyclin-dependent kinases (CDKs) by reconstructing their closest experimentally characterized cyclin-independent ancestor. Using available crystal structures of CDK2, regulatory switches are identified and four possible hypotheses describing why CDK2 requires an extra intra-domain regulatory switch compared to the ancestor are formulated. Each hypothesis is tested using all-atom molecular dynamics simulations. Both systems show similar stability in the K33-E51 hydrogen bond and in the alignment of residues in the regulatory-spine, two key protein kinase regulatory elements, while auto-inhibition due to a helical turn in the a-loop is less favorable in the ancestor. The aspartate of the DFG motif does not form a bidentate bond with Mg in CDK2, unlike the ancestor. Using the results of hypothesizes testing, a set of mutations responsible for the changes in CDK2 are identified. Our findings provide a mechanistic rationale for how evolution has added a new regulatory switch to CDK proteins. Moreover, our approach is directly applicable to other proteins.

evolutionary biology↗

bioRxiv2018

Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus-2 Release from Cells

Herpes simplex virus-2 (HSV-2) can productively infect many different cell types of human and non-human origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L in HSV-2 release from cells. In vaginal epithelial cells and other cell lines tested, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-{kappa}B nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface, and thus facilitate virus release from cells.\n\nImportanceGenital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions, and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-{kappa}B, HPSE is also regulated post-translationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

microbiology↗