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Lung cancer is the most frequent cause of cancer-related deaths worldwide, and mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are a common cause of non-small-cell lung cancers, which is a major subtype of lung cancers. Recently, a series of 5-methylpyrimidine-pyridinone derivatives have been designed and synthesized as novel selective inhibitors of EGFR and EGFR mutants. However, the binding-based inhibition mechanism has not yet been determined. In this study, we carried out molecular dynamic simulations and free-energy calculations for EGFR derivatives to fill this gap. Based on the investigation, the three factors that influence the inhibitory effect of inhibitors are as follows: (1) The substitution site of the Cl atom is the main factor influencing the activity through steric effect; (2) The secondary factors are repulsion between the F atom (present in the inhibitor) and Glu762, and the blocking effect of Lys745 on the phenyl ring of the inhibitor. (3) The two factors function synergistically to influence the inhibitory capacity of the inhibitor. The theoretical results of this study can provide further insights that will aid the design of oncogenic EGFR inhibitors with high selectivity., Competing Interests: The authors declare no conflict of interest.

Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein . Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking., Competing Interests: The authors declare no conflict of interest.

β-lactamases are the main molecules responsible for giving bacterial resistance against β-lactam antibiotics. The study of β-lactamases has allowed the development of antibiotics capable of inhibiting these enzymes. In this context, extended spectrum β-lactamase (ESBL) TLA-1 has spread in Escherichia coli and Enterobacter cloacae clinical isolates during the last 30 years in Mexico. In this research, the 3D structures of ESBL TLA-1 and TLA-1 S70G mutant, both ligand-free and in complex with clavulanic acid were determined by X-ray crystallography. Four clavulanic acid molecules were found in the structure of TLA-1, two of those were intermediaries of the acylation process and were localized covalently bound to two different amino acid residues, Ser70 and Ser237. The coordinates of TLA-1 in complex with clavulanic acid shows the existence of a second acylation site, additional to Ser70, which might be extendable to several members of the subclass A β-lactamases family. This is the first time that two serines involved in binding clavulanic acid has been reported and described to an atomic level., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Brain cholesterol homeostasis is altered in Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of a CAG nucleotide repeat in the HTT gene. Genes involved in the synthesis of cholesterol and fatty acids were shown to be downregulated shortly after the expression of mutant huntingtin (mHTT) in inducible HD cells. Nuclear levels of the transcription factors that regulate lipid biogenesis, the sterol regulatory element-binding proteins (SREBP1 and SREBP2), were found to be decreased in HD models compared to wild-type, but the underlying causes were not known. SREBPs are synthesized as inactive endoplasmic reticulum-localized precursors. Their mature forms (mSREBPs) are generated upon transport of the SREBP precursors to the Golgi and proteolytic cleavage, and are rapidly imported into the nucleus by binding to importin β. We show that, although SREBP2 processing into mSREBP2 is not affected in YAC128 HD mice, mSREBP2 is mislocalized to the cytoplasm. Chimeric mSREBP2-and mSREBP1-EGFP proteins are also mislocalized to the cytoplasm in immortalized striatal cells expressing mHTT, in YAC128 neurons and in fibroblasts from HD patients. We further show that mHTT binds to the SREBP2/importin β complex required for nuclear import and sequesters it in the cytoplasm. As a result, HD cells fail to upregulate cholesterogenic genes under sterol-depleted conditions. These findings provide mechanistic insight into the downregulation of genes involved in the synthesis of cholesterol and fatty acids in HD models, and have potential implications for other pathways modulated by SREBPs, including autophagy and excitotoxicity., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that transforms primary B lymphocytes, yielding lymphoblastoid cell lines (LCLs). EBV-encoded nuclear antigen 2 (EBNA2) and EBV-encoded nuclear antigen leader protein (EBNALP) are the first viral products expressed after EBV infection of primary B lymphocytes and are essential for EBV-induced B-lymphocyte growth transformation. EBNA2 functions as a transcriptional activator of viral and cellular genes, with EBNALP as a coactivator for EBNA2-mediated transcriptional activation. We previously reported that mutant EBNALP with a C-terminal 10-amino-acid truncation loses the ability to coactivate, and has a dominant-negative effect on wild-type-EBNALP-mediated coactivation. However, the functional relevance of EBNALP in maintenance of LCL cell growth has not been investigated. To address this, we have constructed LCL-derived cell clones in which this dominant-negative form of EBNALP (DNLP) is conditionally expressed by the Cre-loxP system. We used these cells to evaluate the effect of DNLP expression on EBV-induced cell proliferation. After drug treatment, the DNLP-expressing LCL clones showed reduced cell proliferation and viability. These results indicate that EBNALP is critical for maintaining LCL growth and EBV-induced cell proliferation.

In the present study, we investigated whether mutant huntingtin (mHTT) impairs mitochondrial functions in human striatal neurons derived from induced pluripotent stem cells (iPSCs). Striatal neurons and astrocytes derived from iPSCs from unaffected individuals (Ctrl) and Huntington's disease (HD) patients with HTT gene containing increased number of CAG repeats were used to assess the effect of mHTT on bioenergetics and mitochondrial superoxide anion production. The human neurons were thoroughly characterized and shown to express MAP2, DARPP32, GABA, synapsin, and PSD95. In human neurons and astrocytes expressing mHTT, the ratio of mHTT to wild-type huntingtin (HTT) was 1:1. The human neurons were excitable and could generate action potentials, confirming successful conversion of iPSCs into functional neurons. The neurons and astrocytes from Ctrl individuals and HD patients had similar levels of ADP and ATP and comparable respiratory and glycolytic activities. The mitochondrial mass, mitochondrial membrane potential, and superoxide anion production in human neurons appeared to be similar regardless of mHTT presence. The present results are in line with the results obtained in our previous studies with isolated brain mitochondria and cultured striatal neurons from YAC128 and R6/2 mice, in which we demonstrated that mutant huntingtin at early stages of HD pathology does not deteriorate mitochondrial functions. Overall, our results argue against bioenergetic deficits as a factor in HD pathogenesis and suggest that other detrimental processes might be more relevant to the development of HD pathology.

Dynamic nature of structural segments is a key modulator of protein's intrinsic stability. Mutants of superoxide dismutase 1 (SOD1) protein, like SOD1 G85R and SOD1 G93A , adopt misfolded states that undergo aggregation in motor neuron cells. In this study, we had used correlative computational studies to investigate the temporal flux of structural alterations in SOD1 G85R and SOD1 G93A that increased the instability and aggregation tendency of these proteins. Molecular dynamics simulation studies showed that the G85R and G93A mutations caused localized transitions of the edge-strands of beta sheets to disordered structures near the mutation regions. Though this structural perturbation did not alter the conformation of the catalytic zinc and copper binding residues, it could dislocate the electrostatic loop of SOD1 G85R . This had rendered the electrostatic loop of SOD1 G85R to be incapable of guiding the substrate to the catalytic cleft. The beta sheet-to-disorder transitions near the mutations had caused the induction of steric clashes in the edge-strand residues, resulting in the loss of several intra-molecular interactions in the mutant SOD1 proteins. These had effected in local structural destabilization and increased aggregation potential in SOD1 G85R and SOD1 G93A . Mutant SOD1 proteins adopted energetically less favorable states, with some changes in the residue-level conformation entropy and solvent-exposed surfaces of the mutation neighboring residues. Collectively, our study demonstrated that the two mutations, G85R and G93A, did not have global effects in changing the SOD1 structure. Instead, the instability-associated aggregation of these mutants arose due to the local structural alterations in the edge-strands of specific beta sheets.Communicated by Ramaswamy H. Sarma.

Potato (Solanum tuberosum) mutant (ST) lacking one isoform of manganese-stabilizing protein (MSPI) of photosystem II exhibited besides spontaneous tuberization also growth changes with strongly impaired root system development. Previous studies revealed marked changes in carbohydrate levels and allocation within ST plant body. To verify causal relationship between changed carbohydrate balance and root growth restriction we engaged dark grown sucrose-supplied root organ-cultures of ST plants to exclude/confirm shoot effects. Unexpectedly, in ST root cultures we observed large alterations in growth and architecture as well as saccharide status similar to those found in the intact plant roots. The gene expression analysis, however, proved PsbO1 transcript (coding MSPI protein) neither in ST nor in WT root-organ cultures. Therefore, the results point to indirect effects of PsbO1 allele absence connected possibly with some epigenetic modulations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Aryl Hydrocarbon Receptor (AhR) is a key player to regulate the expression of a group of enzymes known as cytochrome P450s (CYPs) super family (CYP1A1, CYP1B1, CYP2B6, and CYP2E1) which metabolites diverse endogenous as well as toxic compounds such as Benzo[a] Pyrene (B[a] P) and TCDD. B[a] P induces oxidative stress and causes degeneration of dopaminergic neurons in the midbrain, may leads to Parkinson's disease (PD). The metabolism of B[a] P through the expression of CYPs is mainly triggered after binding of B[a] P within ligand binding domain of AhR. But, the molecular mechanism of AhR mediated xenobiotic metabolism in presence of diverse phytochemicals is yet to be studied. The solved AhR (PDB ID: 5NJ8, 23-273aa) structure lacks information for ligand binding domain therefore both wild type and mutant models were predicted and screened virtually against sixty one natural compounds. The result proposed withaferin A, withanolide A, withanolide B, withanolide D and withanone of plant Withania Somnifera as efficient ligand against both wild type and mutants (V381A and V381D) AhR models. However, in silico studies hypothesised withanolide A as a potent phytochemical to trigger the AhR mediated gene regulation activity of CYPs. The in vivo study in zebra fish model proposed about the neuro protective role of W. Somnifera leaf extract in presence of B[a]P. The present study would throw lights on the molecular mechanism of phytochemicals mediated AhR activity which may be useful in treatment of PD. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Polyhydroxyalkanoates are gaining importance due to their biodegradable nature and close analogy to plastics. Polyhydroxybutyrate (PHB) is the most widely used bioplastic from polyalkanoate family, which is produced by a legion of bacterial species via phbCAB operon encoding β-ketothiolase (PhaA), NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC). Augmentation in the activity of these enzymes is promising for increased PHB production which is achieved by enzyme engineering strategies including non-structural and structural approaches. Our study is deployed on directed evolution-based experimentally reported mutants of PhaB enzyme with increased efficiency due to impact on critical structural factors. We have analyzed and compared the native PhaB with two of its variants Q47L and T173S in complex with their cofactor i.e. NADPH as well as the substrate i.e. acetoacetyl-CoA, via long range molecular dynamics simulations. Interaction profile, MMPBSA, essential dynamics, and free energy landscape analysis revealed that the enzyme efficiency is critically affected by cofactor interactions. It was also observed that mutants have higher equilibrium constant with lesser but optimal affinity for substrate and cofactor than the wild type, which might be the reason for increased efficiency of the mutants via enhanced substrate and cofactor exchange rate. Our study provides insights into the cofactor and substrate binding affinities to PhaB enzyme at atomistic level, which will facilitate designing of highly efficient PhaB enzymes for increased PHB production. Communicated by Ramaswamy H. Sarma.

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single-molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.

Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that controls the function and stability of a wide range of cellular client proteins. Fibronectin (FN) is an extracellular client protein of HSP90, and exogenous HSP90 or inhibitors of HSP90 alter the morphology of the extracellular matrix. Here, we further characterized the HSP90 and FN interaction. FN bound to the M domain of HSP90 and interacted with both the open and closed HSP90 conformations; and the interaction was reduced in the presence of sodium molybdate. HSP90 interacted with the N-terminal regions of FN, which are known to be important for matrix assembly. The highest affinity interaction was with the 30-kDa (heparin-binding) FN fragment, which also showed the greatest colocalization in cells and accommodated both HSP90 and heparin in the complex. The strength of interaction with HSP90 was influenced by the inherent stability of the FN fragments, together with the type of motif, where HSP90 preferentially bound the type-I FN repeat over the type-II repeat. Exogenous extracellular HSP90 led to increased incorporation of both full-length and 70-kDa fragments of FN into fibrils. Together, our data suggested that HSP90 may regulate FN matrix assembly through its interaction with N-terminal FN fragments., Competing Interests: The authors declare no conflicts of interest.

Epigenetic changes are increasingly being appreciated as key events in breast cancer progression. However, breast cancer subtype-specific epigenetic regulation remains poorly investigated. Here we report that EZH2 is a leading candidate of epigenetic modulators associated with the TNBC subtype and that it predicts poor overall survival in TNBC patients. We demonstrate that specific pharmacological or genetic inhibition of EZH2 catalytic activity impairs distant metastasis. We further define a specific EZH2 high population with enhanced invasion, mammosphere formation, and metastatic potential that exhibits marked sensitivity to EZH2 inhibition. Mechanistically, EZH2 inhibition differentiates EZH2 high basal cells to a luminal-like phenotype by derepressing GATA3 and renders them sensitive to endocrine therapy. Furthermore, dissection of human TNBC heterogeneity shows that EZH2 high basal-like 1 and mesenchymal subtypes have exquisite sensitivity to EZH2 inhibition compared with the EZH2 low luminal androgen receptor subtype. These preclinical findings provide a rationale for clinical development of EZH2 as a targeted therapy against TNBC metastasis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Zinc-finger antiviral protein (ZAP) is a host antiviral factor that specifically restricts a wide range of viruses. ZAP selectively binds to CG-dinucleotide-enriched RNA sequences and recruits multiple RNA degradation machines to degrade target viral RNA. However, the molecular mechanism and structural basis for ZAP recognition of specific RNA are not clear. Here, we report the crystal structure of the ZAP N-terminal domain bound to a CG-rich single-stranded RNA, providing the molecular basis for its specific recognition of a CG dinucleotide and additional guanine and cytosine. The four zinc fingers of ZAP adopt a unique architecture and form extensive interactions with RNA. Mutations of both protein and RNA at the RNA-ZAP interacting surface reduce the in vitro binding affinity and cellular antiviral activity. This work reveals the molecular mechanism of ZAP recognition of specific target RNA and also provides insights into the mechanism by which ZAP coordinates downstream RNA degradation processes., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder that prominently affects the basal ganglia, leading to affective, cognitive, behavioral, and motor decline. The primary site of neuron loss in HD is the striatal part of the basal ganglia, with GABAergic medium size spiny neurons (MSNs) being nearly completely lost in advanced HD., Objective: Based on the hypothesis that mutant huntingtin (mHTT) protein injures neurons via transcriptional dysregulation, we set out to establish a transcriptional profile of HD disease progression in the well characterized transgenic mouse model, R6/2, and two Knock-in models (KI); zQ175KI (expressing mutant mouse/human chimeric Htt protein) and HdhQ200 HET KI (carrying one allele of expanded mouse CAG repeats)., Methods: In this study, we used quantitative PCR (qPCR) to evaluate striatal mRNA levels of markers of neurotransmission, neuroinflammation, and energy metabolism., Results: After analyzing and comparing transcripts from pre-symptomatic and symptomatic stages, markers expressed in the basal ganglia MSNs, which are typically involved in maintaining normal neurotransmission, showed a genotype-specific decrease in mRNA expression in a pattern consistent with human studies. In contrast, transcripts associated with neuroinflammation and energy metabolism were mostly unaffected in these animal models of HD., Conclusion: Our results show that transcripts linked to neurotransmission are significantly reduced and are consistent with disease progression in both zQ175KI and R6/2 transgenic mouse models.

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough. Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX). Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering. Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91. Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.

TP53 mutations are found in 50% of all cancers and mutated TP53 status is considered poor for treatment. However, some TP53 mutations exhibit only partial loss-of-function (LOF), meaning they retain residual transcriptional and non-transcriptional activities that are potentially beneficial for therapy. Earlier we have characterized a knock-in mouse model for the partial LOF mutant Trp53 E177R (p53RR). Reduced DNA binding cooperativity of this mutant led to the loss of p53-dependent apoptosis, while p53 functions in cell cycle control, senescence, metabolism, and antioxidant defense remained intact. Concomitantly, tumor suppression was evident but strongly compromised compared to wild-type mice. Here we used the Trp53 E177R mouse as a model to investigate whether residual functions of mutant p53 can be engaged to induce cell death, which is considered the most desirable outcome of tumor therapy. We made use of Mdm2 knock-out in developing embryos as a sensitive tool for detecting remaining p53 activities. Genetic ablation of Mdm2 led to embryonic lethality in Trp53 E177R/E177R homozygotes at days 9.5-11.5. This effect was not rescued by concomitant p21-knockout, indicating its independence of p21-mediated cell cycle arrest. Instead, immunohistochemical analysis showed widespread apoptosis in tissues of defective embryos accompanied by persistent accumulation of p53RR protein. This led to partial restoration of the mutant's proficiency in transcriptional induction of the pro-apoptotic genes Bbc3 (Puma) and Bax . These data indicate that increased quantity can compensate for qualitative defects of p53 mutants and suggest that Mdm2-targeting (potentially in combination with other drugs) might be effective against cells bearing p53 partial LOF mutants.

The cold-adapted alpha-amylase (PHA) from Pseudoalteromonas haloplanktis is a psychrophilic enzyme which demonstrates high activity at low temperatures, but poor thermostability. Most of the method only employed the crystal structure to design the target protein. However, the trajectory of protein molecular dynamics (MD) simulation contained clues about the protein stability. In this study, we combined MD simulation and energy optimization methods to design mutations located at non-conserved residues. Two single point mutants (S255K, S340P) and one integrated mutant (S255K/S340P) enhanced thermostability without affecting the optimal catalytic activity. After incubation at 40 °C for 80 min, the residual activities of mutants S255K, S340P and S255K/S340P were 1.6-, 2.4-, and 2.6-fold greater than that of the wild type (WT). Additionally, the catalytic efficiency values (k cat /K m ) of S255K, S340P, and S255K/S340P also increased 1.9-, 2.0-, and 2.7-fold when compared to WT., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have recently derived a β-N-acetylhexosaminidase, BbhI, from Bifidobacterium bifidum JCM 1254, which could regioselectively synthesize GlcNAcβ1-3Galβ1-4Glc with a yield of 44.9%. Here, directed evolution of BbhI by domain-targeted mutagenesis was carried out. Firstly, the GH20 domain was selected for random mutagenesis using MEGAWHOP method and a small library of 1300 clones was created. A total of 734 colonies with reduced hydrolytic activity were isolated, and three mutants with elevated transglycosylation yields, GlcNAcβ1-3Galβ1-4Glc yields of 68.5%, 74.7%, and 81.1%, respectively, were obtained. Subsequently, nineteen independent mutants were constructed according to all the mutation sites in these three mutants. After transglycosylation analysis, Asp714 and Trp773 were identified as key residues for improvement in transglycosylation ability and were chosen for the second round of directed evolution by site-saturation mutagenesis. Two most efficient mutants D714T and W773R that acted as trans-β-N-acetylhexosaminidase were finally achieved. D714T with the substitution at the putative nucleophile assistant residue Asp714 by threonine showed high yield of 84.7% with unobserved hydrolysis towards transglycosylation product. W773R with arginine substitution at Trp773 residue locating at the entrance of catalytic cavity led to the yield up to 81.8%. The k cat /K m values of D714T and W773R for hydrolysis of pNP-β-GlcNAc displayed drastic decreases. NMR investigation of protein-substrate interaction revealed an invariable mode of the catalytic cavity of D714T, W773R, and WT BbhI. The collective motions of protein model showed the mutations Thr714 and Arg773 exerted little effect on the dynamics of the inside but a large effect on the dynamics of the outside of catalytic cavity.

Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. Its wild-type form was recently implicated in mammalian malignancies of hematologic and non-hematologic tissue origins. Moreover, it was recently identified as a mutated gene in human cancers of various origins. In this review we focus on the functional activities of several of the Vav1 mutants analyzed for their tumorigenic properties. We also discuss the relationship of the tested biochemical properties of Vav1 mutants, E59K, D517E and L801P, to their computer-based predicted properties. These comparisons further enhance the need for integration of computation-based structural analyses with experimental data in order to fully appreciate the activity of mutant proteins. Our comprehensive evaluation supports the classification of Vav1 as a bona fide oncogene in human cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Although the involvement of individual microRNA and lncRNA in the regulation of p21 expression has largely been evidenced, less is known about the roles of functional interactions between miRNAs and lncRNAs in p21 expression. Our previous work demonstrated that miR-509- 3-5p could block cancer cell growth., Methods: To gain an insight into the role of miR-509-3-5p in the regulation of p21 expression, we performed in silico prediction and showed that miR-509-3-5p might target the NONHSAT112228.2, a sense-overlapping lncRNA transcribed by a non-code gene overlapping with p21 gene. Mutation and luciferase report analysis suggested that miR-509-3-5p could target NONHSAT112228.2, thereby blocking its expression. Consistently, NONHSAT112228.2 expression was inversely correlated with both miR-509-3-5p and p21 expression in cancer cells. Ectopic expression of miR-509-3-5p and knockdown of NONHSAT112228.2 both promoted proliferation and migration of cancer cells., Results: Interestingly, high-expression of NONHSAT112228.2 accompanied by low-expression of p21 was observed in lung cancer tissues and associated with lower overall survival., Conclusion: Taken together, our study found a new regulatory pathway of p21, in which MiR-509-3-5p functionally interacts with NONHSAT112228.2 to release p21 expression. MiR-509-3-5p- NONHSAT112228.2 regulatory axis can inhibit the proliferation and migration of lung cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the trinucleotide CAG in the HD gene. While the presence of nuclear aggregates of mutant huntingtin (mHtt) in neurons is a hallmark of HD, the reason behind its toxicity remains elusive., Objective: The present study was conducted to assess a correlation between the number of mHtt aggregates and the severity of HD symptoms in R6/1 mice., Methods: We investigated correlations between behavioral deficits and the level of nuclear mHtt aggregates in different neuroanatomical regions in 3-month-old R6/1 mice, the age at which a large variability of symptom severity between animals has been observed., Results: R6/1 mice were deficient in instinctive and anxiety related behaviors as well as long-term memory capabilities. Significant differences were also found between the sexes; female transgenic mice displayed less severe deficits than males. While the level of mHtt aggregates was correlated with the severity of HD phenotypes in most regions of interest, an opposite relationship also was found for some other regions examined., Conclusions: The obtained results suggest harmful and region-specific roles of mHtt aggregates in HD symptoms.

Congenital insensitivity to pain (OMIM 243000) is an extremely rare disorder caused by loss-of-function mutations in SCN9A encoding Nav1.7. Although the SCN9A mutations and phenotypes of painlessness and anosmia/hyposmia in patients are previously well documented, the complex relationship between genotype and phenotype of congenital insensitivity to pain remains unclear. Here, we report a congenital insensitivity to pain patient with novel SCN9A mutations. Functional significance of novel SCN9A mutations was assessed in HEK293 cells expressing Nav1.7, the results showed that p.Arg99His significantly decreased current density and reduced total Nav1.7 protein levels, whereas p.Trp917Gly almost abolished Nav1.7 sodium current without affecting its protein expression. These revealed that mutations in Nav1.7 in this congenital insensitivity to pain patient still retained partial channel function, but the patient showed completely painlessness, the unexpected genotypic-phenotypic relationship of SCN9A mutations in our patient may challenge the previous findings "Nav1.7 total loss-of-function leads to painlessness." Additionally, these findings are helpful for understanding the critical amino acid for maintaining function of Nav1.7, thus contributing to the development of Nav1.7-targeted analgesics.

Purpose: The Grm6 nob8 mouse carries a missense mutation in the Grm6 gene (p.Met66Leu), and exhibits a reduced b-wave of the electroretinogram (ERG), abnormal localization of metabotropic glutamate receptor 6 (mGluR6) to the depolarizing bipolar cell (DBC) soma, and a reduced level of mGluR6 at the DBC dendritic tips. Although the underlying mechanism remains unknown, one possible explanation is that DBCs cannot efficiently traffic the mutant mGluR6. In that scenario, reducing the total amount of mutant mGluR6 protein might normalize localization, and thus, improve the ERG phenotype as well. The second purpose of this study was to determine whether the abnormal cellular distribution of mutant mGluR6 in Grm6 nob8 retinas might induce late onset DBC degeneration., Methods: We crossed Grm6 nob8 animals with Grm6 nob3 mice, which carry a null mutation in Grm6 , to generate Grm6 nob3/nob8 compound heterozygotes. We used western blotting to measure the total mGluR6 content, and immunohistochemistry to document mGluR6 localization within DBCs. In addition, we examined outer retinal function with ERG and retinal architecture in vivo with spectral domain optical coherence tomography (SD-OCT)., Results: The retinal content of mGluR6 was reduced in the retinas of the Grm6 nob3/nob8 compound heterozygotes compared to the Grm6 nob8 homozygotes. The cellular distribution of mGluR6 in the Grm6 nob3/nob8 compound heterozygotes matched that of the Grm6 nob8 homozygotes, with extensive expression throughout the DBC cell body and limited expression at the DBC dendritic tips. The dark-adapted ERG b-waves of the Grm6 nob3/nob8 mice were reduced in comparison to those of the Grm6 nob8 homozygotes at postnatal day 21 and 28. The overall ERG waveforms obtained from 4- through 68-week old Grm6 nob8 mice were in general agreement for dark- and light-adapted conditions. The maximum response and sensitivity of the dark-adapted ERG b-wave did not change statistically significantly with age. SD-OCT revealed the maintained laminar structure of the retina, including a clear inner nuclear layer (INL) at each age examined (from 11 to 57 weeks old), although the INL in the mice older than 39 weeks of age was somewhat thinner than that seen at 11 weeks., Conclusions: Mislocalization of mutant mGluR6 is not normalized by reducing the total mGluR6. Mislocalized mutant mGluR6 does not trigger substantial loss of DBCs., (Copyright © 2019 Molecular Vision.)

Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in Na V 1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in Na V 1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome's control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Mitochondria are essential organelles whose function is upheld by their dynamic nature. This plasticity is mediated by large dynamin-related GTPases, called mitofusins in the case of fusion between two mitochondrial outer membranes. Fusion requires ubiquitylation, attached to K398 in the yeast mitofusin Fzo1, occurring in atypical and conserved forms. Here, modelling located ubiquitylation to α4 of the GTPase domain, a critical helix in Ras-mediated events. Structure-driven analysis revealed a dual role of K398. First, it is required for GTP-dependent dynamic changes of α4. Indeed, mutations designed to restore the conformational switch, in the absence of K398, rescued wild-type-like ubiquitylation on Fzo1 and allowed fusion. Second, K398 is needed for Fzo1 recognition by the pro-fusion factors Cdc48 and Ubp2. Finally, the atypical ubiquitylation pattern is stringently required bilaterally on both involved mitochondria. In contrast, exchange of the conserved pattern with conventional ubiquitin chains was not sufficient for fusion. In sum, α4 lysines from both small and large GTPases could generally have an electrostatic function for membrane interaction, followed by posttranslational modifications, thus driving membrane fusion events., (© 2019 Schuster et al.)

Listeria monocytogenes , a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (Δ menD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five Δ menD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent Δ menD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI ( lmo1576 ). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes RNA-seq transcriptome analysis revealed five genes ( lmo0944 , lmo1575 , lmo1577 , lmo2005 , and lmo2006 ) expressed at a higher level in the Δ ytoI strain than in the wild-type strain, whereas two genes ( lmo1917 and lmo2103 ) demonstrated lower expression in the Δ ytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a Δ ytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes ., (Copyright © 2019 American Society for Microbiology.)

Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Destabilization of prion protein induces a conformational change from normal prion protein (PrP C ) to abnormal prion protein (PrP SC ). Hydrophobic interaction is the main driving force for protein folding, and critically affects the stability and solvability. To examine the importance of the hydrophobic core in the PrP, we chose six amino acids (V176, V180, T183, V210, I215, and Y218) that make up the hydrophobic core at the middle of the H2-H3 bundle. A few pathological mutants of these amino acids have been reported, such as V176G, V180I, T183A, V210I, I215V, and Y218N. We focused on how these pathologic mutations affect the hydrophobic core and thermostability of PrP. For this, we ran a temperature-based replica-exchange molecular dynamics (T-REMD) simulation, with a cumulative simulation time of 28 μs, for extensive ensemble sampling. From the T-REMD ensemble, we calculated the protein folding free energy difference between wild-type and mutant PrP using the thermodynamic integration (TI) method. Our results showed that pathological mutants V176G, T183A, I215V, and Y218N decrease the PrP stability. At the atomic level, we examined the change in pair-wise hydrophobic interactions from valine-valine to valine-isoleucine (and vice versa), which is induced by mutation V180I, V210I (I215V) at the 180 th -210 th (176 th -215 th ) pair. Finally, we investigated the importance of the π-stacking between Y218 and F175.

Autosomal dominant congenital cataract (ADCC), the most common hereditary disease, is a major cause of eye disease in children. Due to its high genetic and clinical heterogeneity, the identification of ADCC-associated gene mutations is essential for the development of molecular therapies. In this study, we examined a four-generation Chinese pedigree with ADCC and identified putative mutations in ADCC candidate genes via next-generation sequencing (NGS) followed by Sanger sequencing. A novel missense mutation in GJA8 (c.T217C) in ADCC patients causes a serine-to-proline substitution at residue 73 of connexin 50 (Cx50); no mutation was found in unaffected family members and unrelated healthy individuals. Functional analysis revealed that this missense mutation disrupts protein function in human lens epithelial cells (HLEpiCs), which fails to form calcium-sensitive hemichannels. Furthermore, mutant Cx50 leads to decreased ROS scavenging by inhibiting G6PD expression and thus induces cell apoptosis via aberrant activation of the unfolded protein response (UPR). In conclusion, we report a novel GJA8 heterozygous mutation in a Chinese family with a vital role in ADCC, broadening the genetic spectrum of this disease.

α-Synuclein misfolding results in the accumulation of amyloid fibrils in Parkinson's disease. Missense protein mutations (e.g. A53T) have been linked to early onset disease. Although α-synuclein interacts with synaptic vesicles in the brain, it is not clear what role they play in the protein aggregation process. Here, we compare the effect of small unilamellar vesicles (lipid composition similar to synaptic vesicles) on wild-type (WT) and A53T α-synuclein aggregation. Using biophysical techniques, we reveal that binding affinity to the vesicles is similar for the two proteins, and both interact with the helix long axis parallel to the membrane surface. Still, the vesicles affect the aggregation of the variants differently: effects on secondary processes such as fragmentation dominate for WT, whereas for A53T, fibril elongation is mostly affected. We speculate that vesicle interactions with aggregate intermediate species, in addition to monomer binding, vary between WT and A53T, resulting in different consequences for amyloid formation., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Arginase is a bimetallic enzyme that utilizes mainly Mn2+ or Co2+ for catalytic function. In human homolog, the substitution of Mn2+ with Co2+ significantly reduces the Km value without affecting the kcat. However, in the Helicobacter pylori counterpart (important for pathogenesis), the kcat increases nearly 4-fold with Co2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co2+ or Mn2+. This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond molecular dynamics simulations studies. The study shows that the difference in binding affinity of Co2+ and Mn2+ ions with the protein is linked to a different positioning of a loop (-122HTAYDSDSKHIHG134-) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co2+- and Mn2+-proteins alters the proportion of protonated His133 via variation in its pKa. This affects the efficiency of proton transfer - an essential step of l-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterized as a taurine specific system. TauA and SsuA are substrate-binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterized its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent from SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters., (© 2019 The Author(s).)

Factor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53's inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics., Competing Interests: The authors declare no conflict of interest.

Protein kinases of the CK1 family can be involved in numerous physiological and pathophysiological processes. Dysregulated expression and/or activity as well as mutation of CK1 isoforms have previously been linked to tumorigenesis. Among all neoplastic diseases, colon and rectal cancer (CRC) represent the fourth leading cause of cancer related deaths. Since mutations in CK1δ previously found in CRC patients exhibited increased oncogenic features, inhibition of CK1δ is supposed to have promising therapeutic potential for tumors, which present overexpression or mutations of this CK1 isoform. Therefore, it is important to develop new small molecule inhibitors exhibiting higher affinity toward CK1δ mutants. In the present study, we first characterized the kinetic properties of CK1δ mutants, which were detected in different tumor entities. Subsequently, we characterized the ability of several newly developed IWP-based inhibitors to inhibit wild type and CK1δ mutants and we furthermore analyzed their effects on growth inhibition of various cultured colon cancer cell lines. Our results indicate, that these compounds represent a promising base for the development of novel CRC therapy concepts.

Background: Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated., Method: In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway., Results: We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-β and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-β response upon rSIV NS1 Y73F and rSIV NS1 S83A infection., Conclusion: We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous T re-2/ B ub2/ C DC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in Caenorhabditis elegans and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-β (IFN-β) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders., Competing Interests: CN, PE, SG, AH, LN, PM, HL, SI, DR, RP No competing interests declared, (© 2019, Nehammer et al.)

ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I-III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS-DnaA complexes. Based on these and other findings, we propose a model for DARS-DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

Xylanases, capable of hydrolyzing xylans which are abundant in nature, have been employed as important biocatalyst in many industrial processes. Xylanases with advantageous properties, especially excellent thermostability, are in high demand in industry. In this study, we aim to improve the thermostability of XynCDBFV, a fungal GH11 xylanase. To achieve this aim, we discovered residues 87-QNSS-90 with pronounced flexibility based on B-factor analysis, identified highly conserved residues 87-RGHT-90 in GH11 xylanases by multiple sequence alignment, and constructed four single mutants by substituting residues from 87 to 90 by site-directed mutagenesis. Temperature stability measurements showed promising enhancement of thermostability for all four single mutants, and the thermal tolerant ability from strong to weak is N88 G, S90 T, S89H, Q87R, XynCDBFV. Four single mutants all retained higher than 50% activities after incubation at the optimal temperature 60℃ for 1 h, while the retained activity for XynCDBFV was only 20.94% at the same condition. N88 G retained greater than 60% residual activity after incubation at 65℃ for 1 h, while the residual activity of XynCDBFV decreased rapidly, losing all activity after 45 min of incubation. Molecular dynamics simulations and structural analysis were applied to explore the heat-resistant mechanisms for mutants: novel hydrogen bonding interaction were discovered and accounted for the improved thermostability. Enzyme activity of the single mutants compromised with their thermostability and combined mutations displayed antagonistic effect due to the closed contact of the mutated residues. This study confirms that combining B-factor analysis and multiple sequence alignment is an effective strategy for obtaining a thermostable enzyme, and the negative findings help to recognize limitations in xylanase engineering for preferable properties., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system., (Copyright © 2019 by the Genetics Society of America.)

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol-AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol-AmB structures at the cell surface, preventing AmB cytotoxicity.

The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, β- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue proteins, His 403 in Wild-type (WT) CMD was replaced with Arg (H403R variant) with site-directed mutagenesis procedures. According to bioinformatics data, Arg 403 in mutant protein is located near Glu 357 as one of the catalytic residues in a manner that they are able to create a medium-range attractive electrostatistic interaction. Structural studies by Far UV-CD showed that this mutation is accompanied by conversion of a small fraction of α-helix to β-form structure. Fluorescence data reveals that, the hydrophobic regions at the surface of protein, as the binding sites for ANS (8-Anilinonaphthalene-1-sulfonic acid) increase in mutant protein, demonstrating relative inflation of H403R variant compared with WT protein. However, the polarity of microenvironment around chromophores did not change upon mutation. Activity measurement in different ranges of pH and temperatures showed that the optimum values of pH and temperature in mutant enzyme is the same as WT enzyme, however; the activity at optimum points increased in H403R variant. It was also revealed that the H403R variant had slightly improved catalytic efficiency for γ-CD. The same value of activation parameters for both protein variants indicates that mutation does not alter the mechanism of catalysis during enzyme-substrate formation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acinetobacter sp. strain LMB-5 can produce a kind of esterase degrading phthalate esters. However, low activity of Est3563 esterase limited its large-scale application. In this study, computer-aided simulation mutagenesis was used to improve the esterase activity with a tightened screening library and enlarged success rate. Two positive mutants, P218R and A242R, were obtained with 2.5 and 2.1 folds higher than the WT Est3563 esterase, with 11.96 ± 0.45 U·mg -1 and 9.90 ± 0.52 U·mg -1 , respectively. With the help of bioinformatics analysis and three-dimensional printing technology, it was found that the mutations could increase the 240-280 residues swing distance and make them deviate from the catalytic pocket. The instability and deviation of these residues on the lid-like structure of the esterase could deteriorate the seal of the binding pocket and expose the active site. Thus, the catalytic efficiency of the mutants became higher. This result demonstrates that the instability and deviation of the lid-like structure could expand the binding pocket of the esterase and enhance the esterase activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)