The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of ⁵²PPQ⁵⁴ residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the ⁵²PPQ⁵⁴ region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.     

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.     

A Conservative Point Mutation in a Dynamic Antigen-binding Loop of Human Immunoglobulin λ6 Light Chain Promotes Pathologic Amyloid Formation

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ∼30 Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.     

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-competent Partially Unfolded Form

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen–deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.     

Activity of Lymphostatin,A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli,is Dependent on a Cysteine Protease Motif

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.     

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-2₁ screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.     

Cross-over Loop Cysteine C152 Acts as an Antioxidant to Maintain the Folding Stability and Deubiquitinase Activity of UCH-L1 Under Oxidative Stress

Redox-dependent inactivation of deubiquitinases (DUBs) is a critical factor for attenuating their DUB activity in response to cellular oxidative stress. Ubiquitin C-terminal hydrolase isoform (UCH-L1) is an important DUB that is highly expressed in human neuronal cells and is implicated in a myriad of human diseases such as neurodegenerative diseases and cancer. Increasing evidence suggests an important role of UCH-L1 in redox regulation and the protection of neuronal cells from oxidative stress. In this study, we examined the molecular basis of how UCH-L1 responds to oxidation in a reversible manner. Using H₂O₂ as a model oxidant, we showed by mass spectrometry that a subset of methionine and cysteine residues, namely (M1, M6, M12, C90, and C152) were more susceptible to oxidation. Spectroscopic analysis showed that oxidation of C90 can lead to profound structural changes in addition to the loss of function. Importantly, we further demonstrated that C152, which is located at the substrate recognition cross-over loop, serves as a reactive oxygen species (ROS) scavenger to protect catalytic C90 from oxidation under moderate oxidative conditions. Hydrogen-deuterium exchange mass spectrometry analysis provided detailed structural mapping of the destabilizing effect of H₂O₂-mediated oxidation, which resulted in global destabilization far beyond the oxidation sites. These perturbations may be responsible for irreversible aggregation when subject to prolonged oxidative stress.     

A metabolomics approach to investigate the proceedings of mitochondrial dysfunction in rats from prediabetes to diabetes

BackgroundDiabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. Methods: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. Results: The most significant metabolic variations in the DM group’s urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. Conclusion: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.     

Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.     

Passive Diffusion of Ciprofloxacin and its Metalloantibiotic: A Computational and Experimental study

Fluoroquinolones (FQ) are antibiotics widely used in clinical practise, but the development of bacterial resistance to these drugs is currently a critical public health problem. In this context, ternary copper complexes of FQ (CuFQPhen) have been studied as a potential alternative. In this study, we compared the passive diffusion across the lipid bilayer of one of the most used FQ, ciprofloxacin (Cpx), and its ternary copper complex, CuCpxPhen, that has shown previous promising results regarding antibacterial activity and membrane partition. A combination of spectroscopic studies and molecular dynamics simulations were used and two different model membranes tested: one composed of anionic phospholipids, and the other composed of zwitterionic phospholipids. The obtained results showed a significantly higher membrane permeabilization activity, larger partition, and a more favourable free energy landscape for the permeation of CuCpxPhen across the membrane, when compared to Cpx. Furthermore, the computational results indicated a more favourable translocation of CuCpxPhen across the anionic membrane, when compared to the zwitterionic one, suggesting a higher specificity towards the former. These findings are important to decipher the influx mechanism of CuFQPhen in bacterial cells, which is crucial for the ultimate use of CuFQPhen complexes as an alternative to FQ to tackle multidrug-resistant bacteria.     

High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl^- with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl^- cotransporter NKCC1 and the K⁺/Cl^- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.     

Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.     

PARP Activity Fine-tunes the DNA Replication Choreography of Chk1-depleted Cells

Checkpoint Kinase 1 (Chk1) prevents DNA damage by adjusting the replication choreography in the face of replication stress. Chk1 depletion provokes slow and asymmetrical fork movement, yet the signals governing such changes remain unclear. We sought to investigate whether poly(ADP-ribose) polymerases (PARPs), key players of the DNA damage response, intervene in the DNA replication of Chk1-depleted cells. We demonstrate that PARP inhibition selectively alleviates the reduced fork elongation rates, without relieving fork asymmetry in Chk1-depleted cells. While the contribution of PARPs to fork elongation is not unprecedented, we found that their role in Chk1-depleted cells extends beyond fork movement. PARP-dependent fork deceleration induced mild dormant origin firing upon Chk1 depletion, augmenting the global rates of DNA synthesis. Thus, we have identified PARPs as novel regulators of replication fork dynamics in Chk1-depleted cells.     

Active and Passive Destabilization of G-Quadruplex DNA by the Telomere POT1-TPP1 Complex

Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.     

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.