A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3.

Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention. Using computational, biochemical and biophysical tools, we delineated the role of all domains, their combinations and the critical phenylalanine residues in regulating HtrA3 activity, oligomerization and specificity. Our findings underline the crucial roles of the N-terminus as well as the PDZ domain in oligomerization and formation of a catalytically competent enzyme, thus providing new insights into its structure-function coordination. Our study also reports an intricate ligand-induced allosteric switch, which redefines the existing hypothesis of HtrA3 activation besides opening up avenues for modulating protease activity favorably through suitable effector molecules.

Dual specificity phosphatase 9: A novel binding partner cum substrate of proapoptotic serine protease HtrA2.

Human high temperature requirement protease A2 (HtrA2) is a trimeric PDZ bearing proapoptotic serine protease, which is involved in various cellular processes and pathologies. Research in the last decade strongly advocates its role as a potential therapeutic target and therefore warrants the need to minutely investigate its mechanism of action, regulation, interactions with other proteins and its binding specificities. In this particular study, we adopted an in silico approach to predict novel interacting partners and/or substrates of HtrA2 by building a peptide library using a binding pattern search. This library was used to look for novel ligand proteins in the human proteome. Thereafter, the putative interaction was validated using biochemical and cell-based studies. In a first, here we report that HtrA2 shows robust interactions with DUSP9 (Dual specificity phosphatase 9) in GST-pulldown and Co-Immunoprecipitation (Co-IP) experiments and cleaves it in vitro. Besides, we also provided a detailed characterization of the interaction interface. Moreover, this study in general provides an efficient, fast and practical method of candidate ligand library screening for exploring the binding properties of HtrA2.

Identification of a distal allosteric ligand binding pocket in HtrA3.

Human HtrA3 (High temperature requirement protease A3) is a trimeric PDZ bearing propapoptotic serine protease, which is involved in various diseases including cancer and pre-eclampsia. Proposed to be a tumor suppressor, its role as a potential therapeutic target is strongly advocated. Therefore, it becomes imperative to gain insights into its mechanism of action and regulation. Allostery is a well-known mechanism of catalytic activation for many HtrA3 homologs, which opens up avenues for manipulating enzyme functions for therapeutic intervention. In our study, through in silico and biochemical approaches, we have reported for the first time that HtrA3 shows allosteric behaviour. We identified a novel selective binding pocket, which triggers conformational reorientations through signal propagation to the distantly situated active-site pocket via the functionally important loop regions. Using molecular docking, simulation studies and biochemical studies we have identified the regulatory movements at and around the active site pocket. Our study is the first one to report a non-classical binding site for HtrA3, which is instrumental for formation of a catalytically efficient orthosteric pocket upon substrate binding.

Synthesis and Evaluation of Ivacaftor Derivatives with Reduced Lipophilicity.

Mutations in the unique ATP-binding cassette anion channel, the cystic fibrosis conductance regulator (CFTR), lead to the inherited fatal disease known as cystic fibrosis (CF). Ivacaftor enhances channel gating of CFTR by stabilizing its open state and has been approved as monotherapy for CF patients with CFTR gating mutations (e.g., G551D) and as part of combination therapy with lumacaftor for CFTR folding mutations (e.g., ΔF508). However, in the latter context, ivacaftor may destabilize folding-rescued ΔF508-CFTR and membrane-associated proteins and attenuate lumacaftor pharmacotherapy. Here, we tested the hypothesis that the high lipophilicity of ivacaftor may contribute to this effect. We describe the synthesis of three glutamic acid ivacaftor derivatives with reduced lipophilicity that bear different charges at neutral pH (compounds 2, 3, 4). In a cellular ion flux assay, all three restored G551D-CFTR channel activity at comparable or better levels than ivacaftor. Furthermore, unlike ivacaftor, compound 3 did not attenuate levels of folding-rescued ΔF508 at the cell surface. Molecular modeling predicts that the increased polarity of compound 3 allows engagement with polar amino acids present in the binding pocket with hydrogen bonding and ionic interactions, which are collectively higher in strength as compared to hydrophobic interactions that stabilize ivacaftor. Overall, the data suggests that reduced lipophilicity may improve the efficacy of this class of CFTR potentiators when used for folding-rescued ΔF508-CFTR.

Women's Experiences With Family Planning Under COVID-19: A Cross-Sectional, Interactive Voice Response Survey in Malawi, Nepal, Niger, and Uganda.

INTRODUCTION: We conducted an assessment in Malawi, Nepal, Niger, and Uganda to document access-related reasons for not using contraceptive methods during the COVID-19 pandemic that led to unintended pregnancies, describe use of modern contraception among women in potential need of contraception compared to before the pandemic, examine method choice, and describe barriers to contraceptive access and use. METHODS: Between December 2020 and May 2021, we conducted an opt-in phone survey with 21,692 women, followed by an outbound survey with 5,124 women who used modern nonpermanent contraceptive methods or who did not want to get pregnant within 2 years but were not using a modern contraceptive method. The surveys examined current behaviors and documented behaviors before the pandemic retrospectively. We used multivariable logistic regression models to examine factors associated with contraceptive use dynamics during COVID-19. RESULTS: Pregnant women surveyed reported that the pandemic had affected their ability to delay or avoid getting pregnant, ranging from 27% in Nepal to 44% in Uganda. The percentage of respondents to the outbound survey using modern contraception decreased during the pandemic in all countries except Niger. Fear of COVID-19 infection was associated with discontinuing modern contraception in Malawi and with not adopting a modern method among nonusers in Niger. Over 79% of surveyed users were using their preferred method. Among nonusers who tried obtaining a method, reasons for nonuse included unavailability of the preferred method or of providers and lack of money; nonusers who wanted a method but did not try to obtain one cited fear of COVID-19 infection. CONCLUSION: We found evidence of surveyed women attributing unintended pregnancies to the pandemic and examples of constraints to contraceptive access and use on the supply and demand side. The effects of the pandemic must be interpreted within the local contraceptive, health system, and epidemiological context.