Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A.

A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 -3.4 Å resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for "alternate access" to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the "alternate access" mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.

Structural Basis of Aquaporin-4 Autoantibody Binding in Neuromyelitis Optica.

Neuromyelitis Optica (NMO) is an autoimmune disease of the central nervous system where pathogenic autoantibodies target the human astrocyte water channel aquaporin-4 causing neurological impairment. Autoantibody binding leads to complement dependent and complement independent cytotoxicity, ultimately resulting in astrocyte death, demyelination, and neuronal loss. Aquaporin-4 assembles in astrocyte plasma membranes as symmetric tetramers or as arrays of tetramers. We report molecular structures of aquaporin-4 alone and bound to Fab fragments from patient-derived NMO autoantibodies using cryogenic electron microscopy. Each antibody binds to epitopes comprised of three extracellular loops of aquaporin-4 with contributions from multiple molecules in the assembly. The structures distinguish between antibodies that bind to the tetrameric form of aquaporin-4, and those targeting higher order orthogonal arrays of tetramers that provide more diverse bridging epitopes.

Kinetic network modeling with molecular simulation inputs: A proton-coupled phosphate symporter.

Phosphate, an essential metabolite involved in numerous cellular functions, is taken up by proton-coupled phosphate transporters of plants and fungi within the major facilitator family. Similar phosphate transporters have been identified across a diverse range of biological entities, including various protozoan parasites linked to human diseases, breast cancer cells with increased phosphate requirements, and osteoclast-like cells engaged in bone resorption. Prior studies have proposed an overview of the functional cycle of a proton-driven phosphate transporter (PiPT), yet a comprehensive understanding of the proposed reaction pathways necessitates a closer examination of each elementary reaction step within an overall kinetic framework. In this work, we leverage kinetic network modeling in conjunction with a "bottom-up" molecular dynamics approach to show how such an approach can characterize the proton-phosphate co-transport behavior of PiPT under different pH and phosphate concentration conditions. In turn, this allows us to reveal the prevailing reaction pathway within a high-affinity phosphate transporter under different experimental conditions and to uncover the molecular origin of the optimal pH condition of this transporter.

Structural basis of prostaglandin efflux by MRP4.

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

Current understanding of frictional dermatoses: A review.

Human skin is continually exposed to internal and external forces, dynamic as well as static. The skin is normally flexible and can resist mechanical trauma due to friction, pressure, vibration, suction and laceration to a considerable degree. However, an excess of these forces can abnormally affect the structure and function of the skin, setting the stage for the development of a skin disorder. Repetitive trauma can cause lichenification, hyperpigmentation, erythema, scaling, fissuring, blisters, ulceration and chronic alterations. Frictional dermatoses is an under-recognised entity with no clear-cut definition and encompasses a variety of terms such as frictional dermatitis, frictional melanosis, frictional pigmentary dermatoses and certain other named entities, many of which are confusing. The authors propose to define frictional dermatoses as 'a group of disorders caused by repetitive trauma to the skin as a result of friction of varied aetiology which can have a wide range of cutaneous manifestations depending on the type of insult.' The exact prevalence of frictional dermatoses as a separate entity is unknown. Authors who conducted this review include a group of dermatologists and post graduate students from various institutions. Literature was reviewed through PubMed, Medscape, Medline, ResearchGate and Google Scholar using the terms 'frictional dermatitis,' 'friction and skin,' 'dermatoses and culture,' 'clothing dermatitis,' 'friction melanosis,' 'PPE induced dermatoses in COVID-19 era,' etc. A total of 122 articles were reviewed and 100 articles among them were shortlisted and included in the study, after removing duplications. The review was followed up with further deliberation which resulted in the formulation of a new definition and classification of frictional dermatoses taking into account the morphology, histopathological characteristics, anatomical region affected and the major predisposing factors. The rising incidence of mechanical dermatoses in the COVID-19 era was also emphasised.

Recent advances in infectious disease research using cryo-electron tomography.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

Smart data collection for CryoEM.

This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.

Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Fun30 and Rtt109 Mediate Epigenetic Regulation of the DNA Damage Response Pathway in C. albicans.

Fun30, an ATP-dependent chromatin remodeler from S. cerevisiae, is known to mediate both regulation of gene expression as well as DNA damage response/repair. The Fun30 from C. albicans has not yet been elucidated. We show that C. albicans Fun30 is functionally homologous to both S. cerevisiae Fun30 and human SMARCAD1. Further, C. albicans Fun30 can mediate double-strand break end resection as well as regulate gene expression. This protein regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathways. The regulation mediated by C. albicans Fun30 is dependent on its ATPase activity. The expression of FUN30, in turn, is regulated by histone H3K56 acetylation catalyzed by Rtt109 and encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild-type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1, and RAD5 expression; ChIP experiments showed that Fun30 but not H3K56ac regulates the expression of these genes in response to oxidative stress. In contrast, upon treatment with MMS, the expression of RAD9 is upregulated, which is modulated by both Fun30 and H3K56 acetylation. Thus, Fun30 and H3K56 acetylation mediate the response to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

Molecular basis of multistep voltage activation in plant two-pore channel 1.

Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge-bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca2+ and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.

Symmetry Reduction in a Hyperpolarization-Activated Homotetrameric Ion Channel.

Plants obtain nutrients from the soil via transmembrane transporters and channels in their root hairs, from which ions radially transport in toward the xylem for distribution across the plant body. We determined structures of the hyperpolarization-activated channel AKT1 from Arabidopsis thaliana, which mediates K+ uptake from the soil into plant roots. These structures of AtAKT1 embedded in lipid nanodiscs show that the channel undergoes a reduction of C4 to C2 symmetry, possibly to regulate its electrical activation.

Platelet-Rich Plasma in Melasma-A Systematic Review.

BACKGROUND: Melasma is a common relapsing hyperpigmentation disorder, which is often difficult to treat. Platelet-rich plasma (PRP) is a novel modality often used to treat acne scars, androgenic alopecia, chronic wounds, and skin rejuvenation. Recently, it has had a promising role in the treatment of melasma. OBJECTIVE: To review the published evidence on the efficacy and safety of PRP in the treatment of melasma. MATERIALS AND METHODS: A systematic review was performed. A meta-analysis could not be performed because of methodological differences across studies and data heterogeneity. RESULTS: Seven studies were fulfilled and analyzed. Most studies used intradermal injections of PRP and have shown significant improvement in melasma. Microneedling mediated delivery of PRP has been tried in melasma with good results. A single study showed no additional benefit of PRP in patients treated with topical tranexamic acid. Another study showed no benefit of intense pulsed light in patients treated with intradermal PRP. CONCLUSION: Platelet-rich plasma inhibits the melanin synthesis through its various components acting through several mechanisms. It demonstrates a moderate grade of recommendation according to the Oxford Center for Evidence-Based Medicine 2011 standards.

Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2.

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT.

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

A Comparative Study of Microneedling Alone Versus Along with Platelet-Rich Plasma in Acne Scars.

CONTEXT: There are several modalities of treating acne scars. The combination of microneedling and platelet-rich plasma (PRP) is a synergistic approach to treat acne scars. AIMS: The aim was to compare the efficacy of microneedling alone versus microneedling with PRP in acne scars. MATERIALS AND METHODS: This was a split face study conducted on 36 patients with acne scars who underwent four sessions of microneedling with PRP on right side and microneedling alone on left side at monthly interval. The total scars with subtypes and Ecchelle D'Evaluation Cliniques des Cicatrices D'Acne (ECCA) score were assessed at baseline and second, fourth, and sixth visits. Visual analog score (VAS) was evaluated by both physicians and patients. STATISTICAL ANALYSIS: The statistical analysis was carried out using Statistical Package for Social Sciences. Paired-t test and Wilcoxon signed rank test were used to compare the results. RESULTS: Mean age was 23.7±3.2 years with 17 male and 19 female patients. The mean total scars on right and left sides declined from 42.14±21.15 to 25.08±14.14 and 43.28+23.08 to 27.17±15.68, respectively, with insignificant differences (P-value = 0.094). ECCA score on right and left sides declined from 88.31±32.78 to 62.92±23.68 and 89.58±32.43 to 66.25±23.89, respectively (P-value = 0.058). VAS evaluated by patient and physician showed maximum improvement at second and third visits, respectively. CONCLUSIONS: This study showed no added advantage of topical application of PRP over microneedling in acne scars.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.