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Cecchetti, Cristina, Nicola J. Scull, Thotegowdanapalya C. Mohan, Yilmaz Alguel, Alexandra M. C. Jones, Alexander D. Cameron i Bernadette Byrne. "Transfer of stabilising mutations between different secondary active transporter families". FEBS Open Bio 11, nr 6 (8.05.2021): 1685–94. http://dx.doi.org/10.1002/2211-5463.13168.
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Dabravolski, Siarhei A., i Yury K. Kavalionak. "The Worldwide Search for the New Mutations in the RNA-Directed RNA Polymerase Domain of SARS-CoV-2". Macedonian Veterinary Review 44, nr 1 (1.03.2021): 87–94. http://dx.doi.org/10.2478/macvetrev-2020-0036.
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Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, responsible for the current pandemic outbreak. In total, 200 genomes of the SARS-CoV-2 strains from four host organisms have been analyzed. To investigate the presence of the new mutations in the RNA-directed RNA Polymerase (RdRp) of SARS-CoV-2, we analyzed sequences isolated from different hosts, with particular emphasis on human isolates. We performed a search for the new mutations of the RdRp proteins and study how those newly identified mutations could influence RdRp protein stability. Our results revealed 25 mutations in Rhinolophus sinicus, 1 in Mustela lutreola, 6 in Homo sapiens, and none in Mus musculus RdRp proteins of the SARS-CoV-2 isolates. We found that P323L is the most common stabilising radical mutation in human isolates. Also, we described several unique mutations, specific for studied hosts. Therefore, our data suggest that new and emerging variants of the SARS-CoV-2 RdRp have to be considered for the development of effective therapeutic agents and treatments.
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Lillebostad, Peder A. G., Arne Raasakka, Silje J. Hjellbrekke, Sudarshan Patil, Trude Røstbø, Hanne Hollås, Siri A. Sakya, Peter D. Szigetvari, Anni Vedeler i Petri Kursula. "Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment". Biomolecules 10, nr 4 (24.04.2020): 660. http://dx.doi.org/10.3390/biom10040660.
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The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure–function studies of both full-length AnxA11 and the disease variants being identified in ALS.
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Kielty, C. M., T. Rantamaki, A. H. Child, C. A. Shuttleworth i L. Peltonen. "Cysteine-to-arginine point mutation in a ‘hybrid’ eight-cysteine domain of FBN1: consequences for fibrillin aggregation and microfibril assembly". Journal of Cell Science 108, nr 3 (1.03.1995): 1317–23. http://dx.doi.org/10.1242/jcs.108.3.1317.
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Mutations in the FBN1 gene encoding the microfibrillar glycoprotein fibrillin cause Marfan syndrome, a relatively common autosomal dominant connective tissue disease. Causative FBN1 mutations appear to be dispersed throughout the coding frame, and to date no predictable genotype: phenotype correlations have emerged. We have identified a point mutation within an eight-cysteine ‘hybrid’ motif of the fibrillin polypeptide which results in the substitution of an arginine for a cysteine, in a patient severely affected in the cardiovascular, skeletal and ocular systems. We have utilised cell cultures from various tissues of this patient to investigate the effects of this mutation on fibrillin expression and deposition, and the consequences in terms of microfibril assembly and organisation. We have established that there is no difference in the expression of normal and mutant alleles, and fibrillin synthesis, secretion and deposition are also normal. However, the rate of fibrillin aggregation is reduced and microfibrillar assemblies are both remarkably scarce and morphologically abnormal. These data clearly demonstrate that the mutated allele interferes with normal assembly, and strongly implicate this particular region of the fibrillin-1 molecule in stabilising microfibrillar assemblies.
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Willows, Jamie, Maryam Al Badi, Chloe Richardson, Noel Edwards, Sarah Rice i John A. Sayer. "Case Report: Investigation and molecular genetic diagnosis of familial hypomagnesaemia: a case report". F1000Research 8 (15.05.2019): 666. http://dx.doi.org/10.12688/f1000research.19006.1.
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Genetic mutations causing familial hypomagnesaemia syndromes are well-recognised. Affected patients can present with severe symptoms of hypomagnesaemia, such as seizures or cardiac arrhythmia. We report an affected child, from a consanguineous family, who presented in the first weeks of life with seizures secondary to hypomagnesaemia, without other associated clinical features. We performed whole exome sequencing in the affected child and segregation analysis within the family, which revealed a novel homozygous missense mutation in TRPM6, which was confirmed as a heterozygous allele in both parents and two younger siblings who had transient hypomagnesaemia. Using in silico modelling, we provide evidence that the missense variant p.(K1098E) in TRPM6 is pathogenic, as it disrupts stabilising TRP domain interactions. Management of familial hypomagnesaemia relies on prompt recognition, early magnesium replacement and lifelong monitoring.
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Willows, Jamie, Maryam Al Badi, Chloe Richardson, Aisha Al Sinani, Noel Edwards, Sarah Rice i John A. Sayer. "Case Report: Investigation and molecular genetic diagnosis of familial hypomagnesaemia". F1000Research 8 (5.12.2019): 666. http://dx.doi.org/10.12688/f1000research.19006.2.
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Genetic mutations causing familial hypomagnesaemia syndromes are well-recognised. Affected patients can present with severe symptoms of hypomagnesaemia, such as seizures or cardiac arrhythmia. We report an affected child, from a consanguineous family, who presented in the first weeks of life with seizures secondary to hypomagnesaemia, without other associated clinical features. We performed whole exome sequencing in the affected child and segregation analysis within the family, which revealed a novel homozygous missense mutation in TRPM6, which was confirmed as a heterozygous allele in both parents and two younger siblings who had transient hypomagnesaemia. Using in silico modelling, we provide evidence that the missense variant p.(K1098E) in TRPM6 is pathogenic, as it disrupts stabilising TRP domain interactions. Management of familial hypomagnesaemia relies on prompt recognition, early magnesium replacement and lifelong monitoring.
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Kachan, Alexandr, i Anatoliy Evtushenkov. "Thermostable mutant variants of Bacillus sp. 406 α-amylase generated by site-directed mutagenesis". Open Life Sciences 8, nr 4 (1.04.2013): 346–56. http://dx.doi.org/10.2478/s11535-013-0142-0.
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AbstractSeveral mutations are known to increase the thermostability of α-amylase of B. licheniformis and other α-amylases. Site-directed mutagenesis was used to introduce similar mutations into the sequence of the α-amylase gene from mesophilic Bacillus sp. 406. The influence of the mutations on thermostability of the enzyme was studied. It was shown that the Gly211Val and Asn192Phe substitutions increased the half-inactivation temperature (Tm) of the enzyme from 51.94±0.45 to 55.51±0.59 and 58.84±0.68°C respectively, in comparison to the wild-type enzyme. The deletion of Arg178-Gly179 (dRG) resulted in an increase of Tm of the α-amylase to 71.7±1.73°C. The stabilising effect of mutations was additive. When combined they increase the Tm of the wild-type amylase by more than 26°C. Thermostability rates of the triple mutant are close to the values which are typical for industrial heat-stable α-amylases, and its ability to degrade starch at 75°C was considerably increased. The present research confirmed that the Gly211Val, Asn192Phe and dRG mutations could play a significant role in thermostabilization of both mesophilic and thermophilic α-amylases.
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Larkin, Sarah, i Niki Karavitaki. "Recent advances in molecular pathology of craniopharyngioma". F1000Research 6 (24.07.2017): 1202. http://dx.doi.org/10.12688/f1000research.11549.1.
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Craniopharyngiomas are rare epithelial tumours arising along the path of the craniopharyngeal duct. Two major histological subtypes have been recognised, the papillary and the adamantinomatous. Craniopharyngiomas remain challenging tumours to manage and are associated with significant morbidities and mortality. Recent advances in the molecular pathology of these neoplasms have identified BRAF mutations in the papillary variant, offering promising options for targeted pharmacological treatment. The involvement of β-catenin and the Wnt pathway in the tumorigenesis of the adamantinomatous subtype has been previously established with the identification of stabilising mutations in exon 3 of CTNNB1. Further understanding of the pathogenesis of this subtype has been facilitated with the use of mouse models and xenograft experiments. It has been proposed that the clusters of cells with upregulated Wnt/β-catenin signalling induce tumour formation in a paracrine manner; the complex interactions occurring between different cell populations need to be further clarified for further expansion of this hypothesis. This review outlines recent key advances in our understanding of the molecular pathology of craniopharyngiomas and discusses some of the challenges that need to be overcome for the development of targeted therapies that will hopefully improve the management and the outcomes of these patients.
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Monteiro, Fátima Liliana, Cecilia Williams i Luisa A. Helguero. "A Systematic Review to Define the Multi-Faceted Role of Lysine Methyltransferase SETD7 in Cancer". Cancers 14, nr 6 (10.03.2022): 1414. http://dx.doi.org/10.3390/cancers14061414.
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Histone–lysine N-methyltransferase SETD7 regulates a variety of cancer-related processes, in a tissue-type and signalling context-dependent manner. To date, there is no consensus regarding SETD7´s biological functions, or potential for cancer diagnostics and therapeutics. In this work, we summarised the literature on SETD7 expression and function in cancer, to identify the contexts where SETD7 expression and targeting can lead to improvements in cancer diagnosis and therapy. The most studied cancers were found to be lung and osteosarcoma followed by colorectal and breast cancers. SETD7 mRNA and/or protein expression in human cancer tissue was evaluated using public databases and/or in-house cohorts, but its prognostic significance remains inconclusive. The most studied cancer-related processes regulated by SETD7 were cell proliferation, apoptosis, epithelial-mesenchymal transition, migration and invasion with special relevance to the pRb/E2F-1 pathway. SETD7 consistently prevented epithelial to mesenchymal transition in different cancer types, and inhibition of its function appears to be associated with improved response to DNA-damaging agents in most of the analysed studies. Stabilising mutations in SETD7 target proteins prevent their methylation or promote other competing post-translational modifications that can override the SETD7 effect. This indicates that a clear discrimination of these mutations and competing signalling pathways must be considered in future functional studies.
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Szczepaniak, Joanna, Cara Press i Colin Kleanthous. "The multifarious roles of Tol-Pal in Gram-negative bacteria". FEMS Microbiology Reviews 44, nr 4 (30.05.2020): 490–506. http://dx.doi.org/10.1093/femsre/fuaa018.
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ABSTRACT In the 1960s several groups reported the isolation and preliminary genetic mapping of Escherichia coli strains tolerant towards the action of colicins. These pioneering studies kick-started two new fields in bacteriology; one centred on how bacteriocins like colicins exploit the Tol (or more commonly Tol-Pal) system to kill bacteria, the other on the physiological role of this cell envelope-spanning assembly. The following half century has seen significant advances in the first of these fields whereas the second has remained elusive, until recently. Here, we review work that begins to shed light on Tol-Pal function in Gram-negative bacteria. What emerges from these studies is that Tol-Pal is an energised system with fundamental, interlinked roles in cell division – coordinating the re-structuring of peptidoglycan at division sites and stabilising the connection between the outer membrane and underlying cell wall. This latter role is achieved by Tol-Pal exploiting the proton motive force to catalyse the accumulation of the outer membrane peptidoglycan associated lipoprotein Pal at division sites while simultaneously mobilising Pal molecules from around the cell. These studies begin to explain the diverse phenotypic outcomes of tol-pal mutations, point to other cell envelope roles Tol-Pal may have and raise many new questions.
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Todaro, Alice M., Tilman M. Hackeng i Elisabetta Castoldi. "Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model". Applied Sciences 11, nr 20 (15.10.2021): 9621. http://dx.doi.org/10.3390/app11209621.
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Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPIα) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0–20 µM MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPIα (activity) levels.
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Balkwill, Graham D., Huw E. L. Williams i Mark S. Searle. "Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria". Organic & Biomolecular Chemistry 5, nr 5 (2007): 832. http://dx.doi.org/10.1039/b616820e.
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Srivastava, Ambuj, Dhanusha Yesudhas, Shandar Ahmad i M. Michael Gromiha. "Deciphering the Role of Residues Involved in Disorder-To-Order Transition Regions in Archaeal tRNA Methyltransferase 5". Genes 12, nr 3 (11.03.2021): 399. http://dx.doi.org/10.3390/genes12030399.
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tRNA methyltransferase 5 (Trm5) enzyme is an S-adenosyl methionine (AdoMet)-dependent methyltransferase which methylates the G37 nucleotide at the N1 atom of the tRNA. The free form of Trm5 enzyme has three intrinsically disordered regions, which are highly flexible and lack stable three-dimensional structures. These regions gain ordered structures upon the complex formation with tRNA, also called disorder-to-order transition (DOT) regions. In this study, we performed molecular dynamics (MD) simulations of archaeal Trm5 in free and complex forms and observed that the DOT residues are highly flexible in free proteins and become stable in complex structures. The energetic contributions show that DOT residues are important for stabilising the complex. The DOT1 and DOT2 are mainly observed to be important for stabilising the complex, while DOT3 is present near the active site to coordinate the interactions between methyl-donating ligands and G37 nucleotides. In addition, mutational studies on the Trm5 complex showed that the wild type is more stable than the G37A tRNA mutant complex. The loss of productive interactions upon G37A mutation drives the AdoMet ligand away from the 37th nucleotide, and Arg145 in DOT3 plays a crucial role in stabilising the ligand, as well as the G37 nucleotide, in the wild-type complex. Further, the overall energetic contribution calculated using MMPBSA corroborates that the wild-type complex has a better affinity between Trm5 and tRNA. Overall, our study reveals that targeting DOT regions for binding could improve the inhibition of Trm5.
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Powell, S. M., V. Christiaens, D. Voulgaraki, J. Waxman, F. Claessens i C. L. Bevan. "Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate." Endocrine-related cancer 11, nr 1 (marzec 2004): 117–30. http://dx.doi.org/10.1677/erc.0.0110117.
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The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.
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Oni, Clare, Mufaddal Moonim, Joana Desterro, Clive Grattan, Natalia Curto-Garcia, Claire Woodley, Monika Ciesielska i in. "UK Single Institution Experience of Mastocytosis: Guy's and St Thomas' NHS Foundation Trust". Blood 134, Supplement_1 (13.11.2019): 2960. http://dx.doi.org/10.1182/blood-2019-130477.
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Introduction: Systemic mastocytosis (SM) is a heterogeneous disorder of neoplastic mast cells ranging from an indolent to aggressive multi-system disease. We registered our UK Mastocytosis Centre of Excellence with the European Competence Network of Mastocytosis (ECNM) in 2005. Our multidisciplinary team (MDT) includes haematologists, haematopathologists, dermatologist, immunologist and molecular diagnosticians. Over 500 patients have been reviewed in our quarterly SM-MDT. We present a large single centre experience of a rare disorder. Method: We have reviewed prospective and retrospectively collected clinical data for 125 adult patients from 2009. The data captures clinical, laboratory, diagnostic results and management. Local patients with a tryptase level of >20ug/L are offered haematology review and bone marrow investigation with KIT D816V mutation analysis using digital PCR. Patients seen for a second opinion have their diagnostic material reviewed. Since 2018, next generation sequencing (NGS) based myeloid gene panels are carried out on patients with advanced SM (AdvSM). Results: Classification of mastocytosis patients: In 125 patients no gender bias was seen: 56 (44.8%) were male and 69 (55.2%) female. Median age of patients with indolent SM (ISM) was 49 years (range 22-78); and for AdvSM: (aggressive SM, SM with an associated haematological neoplasm and mast cell leukaemia) was 64years (range 5-77). The median serum tryptase level was 48.9ug/L for ISM patients (range 4-682) and 120ug/L for AdvSM patients (range 24-854). 112/125 patients had a bone marrow biopsy. 13 patients chose not to have a biopsy. 88% (110/125) of patients had a mast cell disorder. 4% (5/125) had no marrow involvement. 84% (105/125) met WHO criteria for a diagnosis of SM - Figure 1.These were sub-classified: 71/105 (67%) ISM; 2/105 (2%) smouldering SM (SSM); 3/105 (3%) aggressive SM (ASM); 2/105 (2%) mast cell leukaemia (MCL) and 27/105 (26%) SM with an associated hematologic neoplasm (SM-AHN). The most frequent associated haematological neoplasms were MPN and CMML. Lymphomas (n=3) and plasma cell dyscrasias (n=1) were seen patients. In 10/27 (37%) cases of SM-AHN the SM was the initial diagnosis with subsequent development of an AHN. In 5/27(19%) cases the AHN component preceded the SM and in 12/27 (44%) cases the 2 components were co-diagnosed. Mutation status : 24/32 patients with AdvSM were noted to be positive for an activating KIT mutation (D816V, n=23; D816L, n=1). 15/32 AdvSM patients had NGS myeloid gene panel analysis for the SAR panel (SRSF2, ASXL1 and RUNX1) which confers poor prognosis. 4 of the15 patients harboured at least 2 out of 3 SAR mutations. JAK2 V617F mutation was detected in 7 of 8 classical MPN patients and in 1 of 4 patients with MPNu/MDS overlap. 4 patients had a TET2 mutation. 1 patient with mast cell leukaemia had a GATA2 mutation in addition to all 3 in the SAR panel. Cutaneous involvement: 70% (88/125) of patients had skin involvement: 62% (78/125) urticaria pigments, 3% (4/125) telangiectasia macularis eruptiva perstans and 5% (6/125) having non-specific involvement. Skin involvement was more common in ISM (78%) than AdvSM (54% in SM-AHN, none in the 2 patients with MCL). Management : Treatments in ISM are usually for symptom control. Antihistamines, anti-inflammatory agents, anti-leukotriene agents, mast cell stabilising agents, bisphosphonates, proton-pump inhibitors, PUVA and steroids are used. Various cytoreductive therapies have been used in AdvSM (hydroxycarbamide, pegylated interferon-α, cladrabine, cytarabine, azacitadine alone/combination with midostaurin). Patients with SM-AHN have tailored management depending on the disease component that requires treatment. Access to targeted therapies for AdvSM patients in a trial or compassionate use programme has been successful: 7/32 patients with advanced SM have been enrolled into trials (midostaurin 1; avapritinib 6). Conclusions: Our data reflects the heterogeneous nature of the SM classification spectrum clinically and diagnostically. An experienced MDT provides holistic high standard of care within a comprehensive centre for good patient outcomes (Figure 2). Development of a portfolio of novel therapeutic options in multi-centred, international collaborative trials is beneficial to patients and pivotal for research in this rare disorder. Disclosures Cross: Novartis: Consultancy, Research Funding; Incyte: Consultancy. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Radia:Blueprint Medicines: Consultancy; Novartis: Consultancy, Speakers Bureau. OffLabel Disclosure: There are very few medications available in mastocytosis and various off label medications have been used including pegylated interferon alpha.
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Francis, Nimmy, Malaya R. Behera, Kathiresan Natarajan i Rakesh S. Laishram. "Tyrosine phosphorylation controlled poly(A) polymerase I activity regulates general stress response in bacteria". Life Science Alliance 6, nr 3 (19.12.2022): e202101148. http://dx.doi.org/10.26508/lsa.202101148.
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RNA 3′-end polyadenylation that marks transcripts for degradation is implicated in general stress response inEscherichia coli. Yet, the mechanism and regulation of poly(A) polymerase I (PAPI) in stress response are obscure. We show thatpcnB(that encodes PAPI)-null mutation widely stabilises stress response mRNAs and imparts cellular tolerance to multiple stresses, whereas PAPI ectopic expression renders cells stress-sensitive. We demonstrate that there is a substantial loss of PAPI activity on stress exposure that functionally phenocopiespcnB-null mutation stabilising target mRNAs. We identify PAPI tyrosine phosphorylation at the 202 residue (Y202) that is enormously enhanced on stress exposure. This phosphorylation inhibits PAPI polyadenylation activity under stress. Consequentially, PAPI phosphodeficient mutation (tyrosine 202 to phenylalanine, Y202F) fails to stimulate mRNA expression rendering cells stress-sensitive. Bacterial tyrosine kinase Wzc phosphorylates PAPI-Y202 residue, and thatwzc-null mutation renders cells stress-sensitive. Accordingly,wzc-null mutation has no effect on stress sensitivity in the presence ofpcnB-null orpcnB-Y202Fmutation. We also establish that PAPI phosphorylation-dependent stress tolerance mechanism is distinct and operates downstream of the primary stress regulator RpoS.
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Yu, Sihang, Xiaoyu Yan, Rui Tian, Long Xu, Yuanxin Zhao, Liankun Sun i Jing Su. "An Experimentally Induced Mutation in the UBA Domain of p62 Changes the Sensitivity of Cisplatin by Up-Regulating HK2 Localisation on the Mitochondria and Increasing Mitophagy in A2780 Ovarian Cancer Cells". International Journal of Molecular Sciences 22, nr 8 (13.04.2021): 3983. http://dx.doi.org/10.3390/ijms22083983.
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The study of cisplatin sensitivity is the key to the development of ovarian cancer treatment strategies. Mitochondria are one of the main targets of cisplatin, its self-clearing ability plays an important role in determining the fate of ovarian cancer cells. First, we proved that the sensitivity of ovarian cancer cells to cisplatin depends on mitophagy, and p62 acts as a broad autophagy receptor to regulate this process. However, p62′s regulation of mitophagy does not depend on its location on the mitochondria. Our research shows that the mutation of the UBA domain of p62 increases the localisation of HK2 on the mitochondria, thereby increasing the phosphorylated ubiquitin form of parkin, then stabilising the process of mitophagy and ultimately cell survival. Collectively, our results showed that a mutation in the UBA domain of p62 regulates the level of apoptosis stimulated by cisplatin in ovarian cancer.
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Parker, Margaret M., Simina Ticau, James Butler, David Erbe, Madeline Merkel, Emre Aldinc, Gregory Hinkle i Paul Nioi. "Transthyretin-stabilising mutation T119M is not associated with protection against vascular disease or death in the UK Biobank". Amyloid 27, nr 3 (19.05.2020): 184–90. http://dx.doi.org/10.1080/13506129.2020.1758658.
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Rawlings, Andrea E. "Membrane protein engineering to the rescue". Biochemical Society Transactions 46, nr 6 (31.10.2018): 1541–49. http://dx.doi.org/10.1042/bst20180140.
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The inherent hydrophobicity of membrane proteins is a major barrier to membrane protein research and understanding. Their low stability and solubility in aqueous environments coupled with poor expression levels make them a challenging area of research. For many years, the only way of working with membrane proteins was to optimise the environment to suit the protein, through the use of different detergents, solubilising additives, and other adaptations. However, with innovative protein engineering methodologies, the membrane proteins themselves are now being adapted to suit the environment. This mini-review looks at the types of adaptations which are applied to membrane proteins from a variety of different fields, including water solubilising fusion tags, thermostabilising mutation screening, scaffold proteins, stabilising protein chimeras, and isolating water-soluble domains.
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López-Fanjul, C. "The action of evolutionary forces on metric traits". Advances in Animal Biosciences 1, nr 3 (6.01.2011): 532–37. http://dx.doi.org/10.1017/s2040470010005418.
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Fisher's theorem of natural selection implies that the population genetic variance of quasi-neutral traits should be mostly additive. In the case of fitness component traits, however, that variance would be characterised by a substantial contribution from non-additive loci. In parallel, Robertson's theorem states that selection will change the population mean of a trait proportionally to the magnitude of the genetic correlation between that trait and fitness, which should be weak for quasi-neutral traits or strong for the mean fitness components. Drosophila data from inbreeding and artificial selection experiments are discussed within that theoretical framework. In addition, the process of regeneration by mutation of the genetic variance of a quasi-neutral trait (abdominal bristle number) in a Drosophila population initially homozygous at all loci has been analysed. After 485 generations of mutation accumulation, the levels of additive variance found in this population closely approached those commonly observed in laboratory populations. Furthermore, these values, together with previously reported estimates for natural populations, could be jointly explained by a model assuming weak causal stabilising selection.
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Ahmad, Nurul Nadirah, Nor Hafizah Ahmad Kamarudin, Adam Thean Chor Leow i Raja Noor Zaliha Raja Abd Rahman. "The Role of Surface Exposed Lysine in Conformational Stability and Functional Properties of Lipase from Staphylococcus Family". Molecules 25, nr 17 (25.08.2020): 3858. http://dx.doi.org/10.3390/molecules25173858.
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Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.
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Raval, Vishal, Priyanka Sudana i Taraprasad Das. "Idiopathic myelofibrosis causing inflammatory central retinal vein occlusion mimicking frosted branch angiitis". BMJ Case Reports 12, nr 6 (3.06.2019): e229865. http://dx.doi.org/10.1136/bcr-2019-229865.
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Primary idiopathic myelofibrosis is a clonal disorder arising from the neoplastic transformation of early haematopoietic stem cells leading to abnormal fibrous tissue within bone marrow. We present a 51-year-old man complaining of sudden loss of vision in the right eye along with multiple superficial retinal haemorrhages and perivascular infiltration. Fundus fluorescein angiography and optical coherence tomography confirmed the diagnosis of inflammatory central retinal vein occlusion with macular oedema which mimicked frosted branch angiitis. Laboratory tests and bone marrow biopsy confirmed underlying systemic disease to be Janus kinase 2 mutation positive primary idiopathic myelofibrosis. He received one intravitreal injection of antivascular endothelial growth factor along with dexamethasone for his ocular condition. In view of his systemic disease, he was started on systemic chemotherapy and oral corticosteroid which helped in stabilising the eye condition.
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Ling, Sunbin, Qiaonan Shan, Qifan Zhan, Qianwei Ye, Peng Liu, Shengjun Xu, Xin He i in. "USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1α/USP22 positive feedback loop upon TP53 inactivation". Gut 69, nr 7 (27.11.2019): 1322–34. http://dx.doi.org/10.1136/gutjnl-2019-319616.
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ObjectiveWe aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions.DesignCell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC.ResultsUSP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC.ConclusionUSP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.
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Juliano, John Paul, David H. Small i Marie-Isabel Aguilar. "Peptidomimetic Modulators of BACE1". Australian Journal of Chemistry 73, nr 4 (2020): 366. http://dx.doi.org/10.1071/ch19594.
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The β-site APP Cleaving enzyme 1 (BACE1) is a membrane-associated aspartyl protease which mediates the production of amyloid-β (Aβ), a major component of amyloid plaques in the Alzheimer’s disease brain. We have synthesised and characterised a series of peptidomimetic analogues of BACE substrates that incorporate two distinct stabilising structures. To demonstrate the potential activity of these compounds, a variety of assaying strategies were used to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. β-Amino acids and scissile site N-methylation were incorporated into peptide substrate templates as transition state isostere (TSI) substitutes by positional scanning to generate series of non-TSI β-peptidomimetics. The amino acid sequences flanking the β-cleavage site within APP carrying the Swedish double mutation (APPSW), Neuregulin, the synthetic hydroxyethylene-based TSI peptide inhibitor OM99-2, and the high affinity peptide sequence SEISYEVEFR, served as the four substrate templates from which over 60 peptides were designed and synthesised by solid phase peptide synthesis. A quenched fluorescent substrate BACE1 assay in conjunction with liquid chromatography–mass spectrometry (LC-MS) analysis was established to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. It was determined that β-amino acids substituted at the P1 scissile site position within known peptide substrates were resistant to proteolysis, and particular substitutions induced a concentration-dependent stimulation of BACE1, indicating a possible modulatory role of native BACE1 substrates.
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Matsumoto, Rui, Takashi Sugihara, Hideho Wada, Masayoshi Souri i Akitada Ichinose. "Alloantibodies against the B subunit of plasma factor XIII developed in its congenital deficiency". Thrombosis and Haemostasis 109, nr 04 (2013): 661–68. http://dx.doi.org/10.1160/th12-12-0936.
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SummaryFactor XIII (FXIII) is a fibrin-stabilising factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). FXIII-B prevents the fast clearance of FXIII-A from the circulation. Congenital FXIII-A deficiency is a rare bleeding disorder, and congenital FXIII-B deficiency is even rarer. Through our recent nationwide survey on “acquired haemophilia-like disease due to anti-FXIII autoantibodies,” we newly diagnosed severe congenital FXIII-B deficiency in a Japanese man. He developed thrombocytopenia and gingival bleedings at the age of 73, and his FXIII activity was as low as 10% of the normal. When he suddenly developed spontaneous intramuscular haematoma, the bleeding was arrested by infusing FXIII concentrates. However, his FXIII activity remained around 10% of the normal. At the age of 74, ELISA and western blotting assay unexpectedly revealed complete absence of FXIII-B in the patient’s plasma. A dot blot assay detected anti-FXIII-B alloantibodies for the first time in this disease, which could be attributed to the infusion of exogenous FXIII. He was found to be homozygous for a Japanese founder-effect mutation of F13B. Repeated infusions of exogenous FXIII for hemostasis increased anti-FXIII-B alloantibodies that resisted FXIII substitution. To the best knowledge of the authors, none of the remaining 10 reported cases of congenital FXIII-B deficiency developed alloantibodies to exogenous FXIII-B of plasma FXIII. An originally mild bleeding phenotype of severe congenital FXIII-B deficiency can be exaggerated by additional acquired conditions. Physicians should consider congenital FXIII-B deficiency when they encounter cases of unexplained bleeding disorders.
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Michalski, Michal, i Piotr Setny. "Two modes of fusogenic action for influenza virus fusion peptide". PLOS Computational Biology 19, nr 5 (26.05.2023): e1011174. http://dx.doi.org/10.1371/journal.pcbi.1011174.
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The entry of influenza virus into the host cell requires fusion of its lipid envelope with the host membrane. It is catalysed by viral hemagglutinin protein, whose fragments called fusion peptides become inserted into the target bilayer and initiate its merging with the viral membrane. Isolated fusion peptides are already capable of inducing lipid mixing between liposomes. Years of studies indicate that upon membrane binding they form bend helical structure whose degree of opening fluctuates between tightly closed hairpin and an extended boomerang. The actual way in which they initiate fusion remains elusive. In this work we employ atomistic simulations of wild type and fusion inactive W14A mutant of influenza fusion peptides confined between two closely apposed lipid bilayers. We characterise peptide induced membrane perturbation and determine the potential of mean force for the formation of the first fusion intermediate, an interbilayer lipid bridge called stalk. Our results demonstrate two routes through which the peptides can lower free energy barrier towards fusion. The first one assumes peptides capability to adopt transmembrane configuration which subsequently promotes the creation of a stalk-hole complex. The second involves surface bound peptide configuration and proceeds owing to its ability to stabilise stalk by fitting into the region of extreme negative membrane curvature resulting from its formation. In both cases, the active peptide conformation corresponds to tight helical hairpin, whereas extended boomerang geometry appears to be unable to provide favourable thermodynamic effect. The latter observation offers plausible explanation for long known inactivity of boomerang-stabilising W14A mutation.
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Walden, Miriam, John Edwards, Aleksandra Dziewulska, Uli Schwarz-Linek i Mark Banfield. "Covalent host-targeting by thioester domains of Gram-positive pathogens". Acta Crystallographica Section A Foundations and Advances 70, a1 (5.08.2014): C848. http://dx.doi.org/10.1107/s2053273314091517.
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Gram-positive pathogens are a major concern to global health, with increasing resistance to antimicrobials and the lack of preventative therapeutics. Understanding how these bacteria interact with host cells is vital for the development of novel strategies to combat disease. One of the most crucial steps in infection is adhesion to the host cell. The discovery of complex cell-surface associated proteins, such as pili, has advanced our knowledge of this interaction, however the precise molecular mechanisms underlying this process remain unclear. Structural studies of pili revealed the presence of highly unusual intramolecular covalent bonds between amino acid side chains. These include isopeptide bonds between Lys and Asp/Asn residues, conferring mechanical strength, thermal stability and resistance to proteases [1,2]. In Streptococcus pyogenes pili, the adhesin Spy0125 (or Cpa) interacts with the host cell. It comprises three domains, two of which contain stabilising isopeptide bonds [2,3]. Intriguingly, the third domain contains an extremely rare thioester bond, between a Cys and a Gln residue. A Cys to Ala mutation results in a 75% reduction in adhesion, suggesting that this internal linkage may mediate direct attachment [3]. We have now discovered putative thioester domains (TEDs) in cell-surface proteins of several clinically important pathogens. The only other example of an internal thioester is found in complement proteins, where the reactive bond enables the formation of covalent attachment to pathogens. The presence of these bonds in bacterial proteins suggests the possibility of an as-yet uncharacterised, conserved mechanism of covalent host cell attachment. For a selection of pathogens, we have used mass spectrometry and crystallography to confirm the presence of the covalent link between the Cys and Gln residues within the TEDs. Furthermore, we have identified putative host cell targets of TEDs and confirmed covalent linkages between the TED and the target.
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Shaikh, Afroz, Tapas Kundu, Kanjaksha Ghosh, Omkar Kumat i Gulfisha Mirza. "Factor XIII Deficiency Managed With Individualized Homoeopathic Medicine In Intellectually Disabled Child: An Evidence Based Case Report". Homœopathic Links 35, nr 04 (grudzień 2022): 307–14. http://dx.doi.org/10.1055/s-0042-1743479.
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AbstractFactor XIII deficiency is an inherited bleeding disorder, which is rare, ∼1 in 5,000,000, while its combination with an intellectual disability is seldom. It is mostly a result of genetic mutation caused due to consanguineous marriage. Due to deficiency of fibrin stabilising factor, a clot formed is weak and leads to fibrinolysis, which is evident as unstoppable bleeding from umbilical cord stump, epistaxis and bleeding from various organs. The condition is made worst due to intellectual disability as the patient is more prone to injuries due to lack of knowledge of impending dangers. The latest provision of gene therapy is yet to be achieved in such cases and therefore the biggest challenge lies in keeping these patients fit and functional with optimum quality of life. The anxiety of parents is intensified due to the overall condition of the patient. This case report presents homoeopathic management of one such case. The patient was under treatment for almost a decade. Acute homoeopathic medicines efficiently managed acute haemorrhagic episodes. Constitutional medicine Bufo Rana was prescribed in the asymptomatic phase, which helped to reduce the frequency and intensity of bleeding and also helped to improve social quotient of the patient. The social quotient was assessed employing the Vineland Social Maturity Scale, and the result shows that the social quotient of the patient has improved from 8.3 to 41.66. The anxiety of parents also reduced, which was measured using the Family Interview for Stress and Coping in Mental Retardation Scale. The case analysis was done using a paired t-test. The p-value is less than 0.0001 and highly significant. The evaluation of the homoeopathic treatment was done using modified Naranjo criteria for homoeopathy. Homoeopathy medicines efficiently managed the acute episodes of bleeding and also showed improvement in the intellectual disability.
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Sadasivam, Nandini, Mufaddal Moonim, Clive Grattan, Jonathan White, Bridget Wilkins, Jon van Der Walt, Nicholas C. P. Cross, Claire N. Harrison, Jennifer Thomson i Deepti Radia. "UK Single Instituition Experience of Mastocytosis: Guys and St Thomas' NHS Foundation Trust". Blood 116, nr 21 (19.11.2010): 5052. http://dx.doi.org/10.1182/blood.v116.21.5052.5052.
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Abstract Abstract 5052 Introduction: Systemic mastocytosis(SM) is diagnosed when clonal, neoplastic mast cells are demonstrated in extracutaneous tissues. SM is a heterogeneous disorder ranging from indolent disease to aggressive multisystem involvement. We have an established mastocytosis working group in our Trust which was registered with the European Competence Network of Mastocytosis(ECNM) in 2005. We present data prospectively collected using an ECNM algorithm for the management of SM patients over 5 years. Methods: 120 cases of cutaneous mastocytosis have been discussed at 3 monthly multidisciplinary meetings with dermatology colleagues. Full blood counts, liver, bone profile and DEXA scans are reviewed with clinical symptoms and treatments. Patients with a tryptase level of >20ng/ml are offered haematology review and bone marrow investigation. In addition patients referred directly to haematology for a second opinion have their cases and bone marrows reviewed. Bone marrow samples are sent for c-kit D816V mutation analysis. Results: Classification of SM patients. 59/120 (46%) patients were offered bone marrow biopsies. Tryptase levels for these ranged from 15.1–760ng/ml (median 51.5ng/ml).4 patients declined biopsy.5/55 had normal biopsies and were c-kit negative.50 patients had SM.47/50(94%) met the WHO major criteria and 3/50(6%) minor criteria. These were subclassified-38/50(76%) had Indolent Systemic Mastocytosis(ISM);1/50 (2%) had Smouldering Systemic Mastocytosis(SSM);5/50(10%) had Aggressive Systemic Mastocytosis and 6/50(12%) had Systemic Mastocytosis with associated haematologiocal non-mast cell lineage disorder(SM-AHNMD). The bone marrow trephine disease burden was variable:ASM (range 5–100%), SSM 20%, ISM (5-45%) and AHNMD (5-100%). Tryptase levels reflected total disease bulk including cutaneous burden. C-KIT D816V mutation 44 patients with SM had samples analysed for the D816V mutation. 35 were positive (80%) and 9 negative (20%). Clinical Symptoms: 11/59 (19%) patients were asymptomatic (10 had ISM and 1 SSM).36/59 (61%) patients had urticarial symptoms needing symptomatic treatment (2 normal marrow, 2 AHNMD, 2 ASM and 30 ISM).16/59 (27%) patients had allergic symptoms ranging from mild allergies to anaphylaxis.(3 normal marrows, 13 ISM).13/59(22%) had gastrointestinal symptoms ranging from loose motions to severe colitis(1 normal marrow,1 ASM and 11 ISM). DEXA results:36/59(61%) patients had reported DEXA scans at our Trust, the rest reviewed locally. 6/36 (16%) had osteoporosis and required treatment. One 63yr old female patient has SM-AHNMD(MPD). 5 patients had ISM. 3 were females (age range 45–65yrs; tryptase levels 42.9, 49.1 and 60.5ng/ml)) and 2 male (both 45yrs: tryptase levels 31.2 and 47.8ng/ml). After 1 year of treatment with bisphophonates one of the male patients showed an improvement in his osteoporosis indices.7/36(19%) had osteopaenia reported all with a diagnosis of ISM.5 were male and 2 female (tryptase range 21.1–174ng/ml:median 40.3ng/ml).23/36(64%) patients had normal DEXA scans. Management: Treatment regimes in patients with SM are for symptom control. Antihistamines, anti-inflammatory agents, anti-leucotriene agents, mast cells stabilising agents, bisphosphonates and steroids are used. In severe anaphylactic patients self administration of adrenaline is taught. Patients with ASM have been treated with various modalities e.g. Cladrabine, Alpha Interferon, Dasatinib, Imatinib (D816V negative patients) and Midostaurine with variable partial responses.2 patients with ASM have died due to rapidly progressive disease.2 AHNMD patients have an associated MPD (1 Jak 2 postive) and are being treated with pegylated alpha interferon and venesection.2 AHNMD patients died as a result progressive acute myeloid leukaemia and Non Hodgkins Lymphoma.2 AHNMD patients (MDS/MGUS) are being monitored. Conclusions: Our data reflects the heterogeneous nature of this disorder both clinically and in the histological classification. Patients with ISM can have severe clinical manifestations and treatment needs to be tailored to the individual's symptoms. Assessment and surveillence for osteoporosis is vital for all patients. ASM patients have limited treatment options with variable and unsustained responses. Further development of evidence based novel therapies requires multicentred trials in this rare group of patients. Disclosures: Harrison: Incyte: Honoraria; Novartis: Honoraria.
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Irumagawa, Shin, Kaito Kobayashi, Yutaka Saito, Takeshi Miyata, Mitsuo Umetsu, Tomoshi Kameda i Ryoichi Arai. "Rational thermostabilisation of four-helix bundle dimeric de novo proteins". Scientific Reports 11, nr 1 (6.04.2021). http://dx.doi.org/10.1038/s41598-021-86952-2.
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AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
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Martyna, Agnieszka, Helen Fox i Andrew Macadam. "Design of stable Enterovirus 71 virus like particles (VLPs) as a potential approach to vaccine development". Access Microbiology 2, nr 7A (1.07.2020). http://dx.doi.org/10.1099/acmi.ac2020.po0519.
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We have previously described the design of stable and immunogenic polio virus-like particles (VLPs) (Fox, et al. 2017) as an alternative approach to vaccine production. Unlike current polio vaccines, recombinantly-expressed VLP vaccines are non-infectious so would pose no risk of accidental escape from production plants, threatening eradication. To do this we devised a pipeline for the identification of stabilising mutations which could then be combined in a single construct to produce suitable particles; this strategy may have applications for other enterovirus vaccines. Enterovirus 71 (EV71) is one of causative agents of hand, foot and mouth disease which is usually mild but in some cases neurological and systemic complications may occur. Recently there have been several outbreaks with significant mortality in South East Asia as well as increasing numbers of reports of outbreaks in Europe. VLP vaccines might be a useful alternative to inactivated vaccines currently in use or development. EV71, like poliovirus, produces empty particles that are antigenically different from the virion. If, like poliovirus, these empty particles are less immunogenic than the virion, it would be necessary to stabilise them in the native conformation. We are attempting to do this (1) by incorporating modifications that proved successful in the context of poliovirus and (2) by identifying new candidate mutations using an analogous pipeline. Here we will report the characterisation of a range of different modifications that have stabilising and de-stabilising effects on EV71 particles as well as unexpected effects on morphogenesis.
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Gerasimavicius, Lukas, Xin Liu i Joseph A. Marsh. "Identification of pathogenic missense mutations using protein stability predictors". Scientific Reports 10, nr 1 (21.09.2020). http://dx.doi.org/10.1038/s41598-020-72404-w.
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Abstract Attempts at using protein structures to identify disease-causing mutations have been dominated by the idea that most pathogenic mutations are disruptive at a structural level. Therefore, computational stability predictors, which assess whether a mutation is likely to be stabilising or destabilising to protein structure, have been commonly used when evaluating new candidate disease variants, despite not having been developed specifically for this purpose. We therefore tested 13 different stability predictors for their ability to discriminate between pathogenic and putatively benign missense variants. We find that one method, FoldX, significantly outperforms all other predictors in the identification of disease variants. Moreover, we demonstrate that employing predicted absolute energy change scores improves performance of nearly all predictors in distinguishing pathogenic from benign variants. Importantly, however, we observe that the utility of computational stability predictors is highly heterogeneous across different proteins, and that they are all inferior to the best performing variant effect predictors for identifying pathogenic mutations. We suggest that this is largely due to alternate molecular mechanisms other than protein destabilisation underlying many pathogenic mutations. Thus, better ways of incorporating protein structural information and molecular mechanisms into computational variant effect predictors will be required for improved disease variant prioritisation.
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Zhou, Yunzhuo, Qisheng Pan, Douglas E. V. Pires, Carlos H. M. Rodrigues i David B. Ascher. "DDMut: predicting effects of mutations on protein stability using deep learning". Nucleic Acids Research, 7.06.2023. http://dx.doi.org/10.1093/nar/gkad472.
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Abstract Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.
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Usher, Samuel G., Frances M. Ashcroft i Michael C. Puljung. "Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry". eLife 9 (7.01.2020). http://dx.doi.org/10.7554/elife.52775.
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Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.
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Nisha C, Rakesh K. "Engineering of A Lipase towards Thermostability: Studies on Additive Effect of the two Thermo-Stabilising Mutations at Protein Surface". Advancements in Genetic Engineering 04, nr 02 (2015). http://dx.doi.org/10.4172/2169-0111.1000126.
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Hanyroup, Sharika, Ross C. Anderson, Selvaraj Nataraja, Henry N. Yu, Robert P. Millar i Claire L. Newton. "Rescue of cell surface expression and signalling of mutant follicle-stimulating hormone receptors". Endocrinology, 30.06.2021. http://dx.doi.org/10.1210/endocr/bqab134.
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Abstract Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small-molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilising folding and ‘rescuing’ cell surface expression. We previously demonstrated rescue of cell surface expression of luteinising hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells and cell surface expression was determined by ELISA of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for eleven, modestly reduced to 66–84% of WT for four and was not reduced for two. Of the eleven with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for six by treatment with 1 µM CAN1404 for 24 h and a corresponding increase in FSH-induced signalling was observed for four of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR PCs.
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Lee, Weiluo, Subhas Pradhan, Cheng Zhang, Niccolo A. E. Venanzi, Weina Li, Stephen Goldrick i Paul A. Dalby. "Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli". Scientific Reports 12, nr 1 (21.10.2022). http://dx.doi.org/10.1038/s41598-022-22574-6.
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AbstractBovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.
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Sankaran, Bindu Parayil, Sachin Gupta, Michel Tchan, Beena Devanapalli, Yusof Rahman, Peter Procopis i Kaustuv Bhattacharya. "GLRX5-associated [Fe-S] cluster biogenesis disorder: further characterisation of the neurological phenotype and long-term outcome". Orphanet Journal of Rare Diseases 16, nr 1 (3.11.2021). http://dx.doi.org/10.1186/s13023-021-02073-z.
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Abstract Background Identification and characterisation of monogenic causes of complex neurological phenotypes are important for genetic counselling and prognostication. Bi-allelic pathogenic variants in the gene encoding GLRX5, a protein involved in the early steps of Fe-S cluster biogenesis, are rare and cause two distinct phenotypes: isolated sideroblastic anemia and a neurological phenotype with variant non-ketotic hyperglycinemia. In this study, we analysed the evolution of clinical and MRI findings and long-term outcome of patients with GLRX5 mutations. Methods Four patients from three Australian families of Lebanese descent were identified. All patients presented in childhood and were followed up into adult life through multiple clinical assessments. All were prescribed sodium benzoate. Results All patients (all females, age range 18–56 years) showed a complex neurological phenotype characterised by varying combinations of spastic paraparesis, length-dependent motor/sensory-motor axonal polyneuropathy, and psychiatric disturbances with variable intellectual disability. All had non-ketotic hyperglycinemia and a homozygous pathogenic c.151_153delAAG (p.K51del) change in GLRX5. Motor disability gradually progressed reaching moderate disability during adolescence and moderately severe disability during adult life. The major MRI finding was the upper cervical spinal cord signal changes with contrast enhancement noted in all and additional leukoencephalopathy in one. On follow up MRI, the white matter lesions diminished on a subsequent scan and then remained static over time. The spinal cord showed gliotic changes. Two patients have previously demonstrated low pyruvate dehydrogenase complex deficiency but none had plasma lactate elevation, nor biochemical evidence of branch-chain keto-dehydrogenase deficiency. Glycine levels reduced in patients that tolerated sodium benzoate, possibly stabilising clinical manifestations. Conclusions This report demonstrates that the p.K51del GLRX5 variant causes a distinct and predictable neurological phenotype. The clinical assessments spanning from childhood to adult life enable physicians to infer the natural history of GLRX5 related neurological disorder. There may be widespread metabolic consequences, and optimal management is unknown.
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Sandberg, Anders, Ernesto Berenjeno-Correa, Rosa Crespo Rodriguez, Michael Axenhus, Sophia Schedin Weiss, Kevin Batenburg, Jeroen J. M. Hoozemans, Lars O. Tjernberg i Wiep Scheper. "Aβ42 oligomer-specific antibody ALZ-201 reduces the neurotoxicity of Alzheimer’s disease brain extracts". Alzheimer's Research & Therapy 14, nr 1 (29.12.2022). http://dx.doi.org/10.1186/s13195-022-01141-1.
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Abstract Background In Alzheimer’s disease (AD), amyloid-β 1–42 (Aβ42) neurotoxicity stems mostly from its soluble oligomeric aggregates. Studies of such aggregates have been hampered by the lack of oligomer-specific research tools and their intrinsic instability and heterogeneity. Here, we developed a monoclonal antibody with a unique oligomer-specific binding profile (ALZ-201) using oligomer-stabilising technology. Subsequently, we assessed the etiological relevance of the Aβ targeted by ALZ-201 on physiologically derived, toxic Aβ using extracts from post-mortem brains of AD patients and controls in primary mouse neuron cultures. Methods Mice were immunised with stable oligomers derived from the Aβ42 peptide with A21C/A30C mutations (AβCC), and ALZ-201 was developed using hybridoma technology. Specificity for the oligomeric form of the Aβ42CC antigen and Aβ42 was confirmed using ELISA, and non-reactivity against plaques by immunohistochemistry (IHC). The antibody’s potential for cross-protective activity against pathological Aβ was evaluated in brain tissue samples from 10 individuals confirmed as AD (n=7) and non-AD (n=3) with IHC staining for Aβ and phosphorylated tau (p-Tau) aggregates. Brain extracts were prepared and immunodepleted using the positive control 4G8 antibody, ALZ-201 or an isotype control to ALZ-201. Fractions were biochemically characterised, and toxicity assays were performed in primary mouse neuronal cultures using automated high-content microscopy. Results AD brain extracts proved to be more toxic than controls as demonstrated by neuronal loss and morphological determinants (e.g. synapse density and measures of neurite complexity). Immunodepletion using 4G8 reduced Aβ levels in both AD and control samples compared to ALZ-201 or the isotype control, which showed no significant difference. Importantly, despite the differential effect on the total Aβ content, the neuroprotective effects of 4G8 and ALZ-201 immunodepletion were similar, whereas the isotype control showed no effect. Conclusions ALZ-201 depletes a toxic species in post-mortem AD brain extracts causing a positive physiological and protective impact on the integrity and morphology of mouse neurons. Its unique specificity indicates that a low-abundant, soluble Aβ42 oligomer may account for much of the neurotoxicity in AD. This critical attribute identifies the potential of ALZ-201 as a novel drug candidate for achieving a true, clinical therapeutic effect in AD.
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Hosszu, Laszlo L. P., Rebecca Conners, Daljit Sangar, Mark Batchelor, Elizabeth B. Sawyer, Stuart Fisher, Matthew J. Cliff i in. "Structural effects of the highly protective V127 polymorphism on human prion protein". Communications Biology 3, nr 1 (29.07.2020). http://dx.doi.org/10.1038/s42003-020-01126-6.
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AbstractPrion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.
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Garifulina, Aleksandra, Theres Friesacher, Marco Stadler, Eva-Maria Zangerl-Plessl, Margot Ernst, Anna Stary-Weinzinger, Anita Willam i Steffen Hering. "β subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis". Communications Biology 5, nr 1 (3.08.2022). http://dx.doi.org/10.1038/s42003-022-03720-2.
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AbstractGamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.
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Varshney, Dhaval, Sergio Martinez Cuesta, Barbara Herdy, Ummi Binti Abdullah, David Tannahill i Shankar Balasubramanian. "RNA G-quadruplex structures control ribosomal protein production". Scientific Reports 11, nr 1 (23.11.2021). http://dx.doi.org/10.1038/s41598-021-01847-6.
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AbstractFour-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.
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Paul, Franziska Christina, i Andrew Cumbers. "The return of the local state? Failing neoliberalism, remunicipalisation, and the role of the state in advanced capitalism". Environment and Planning A: Economy and Space, 3.11.2021, 0308518X2110504. http://dx.doi.org/10.1177/0308518x211050407.
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Picking up on the manifestation of state intervention following the 2008 financial crisis, we argue that the recent trend towards remunicipalisation underlines but also problematises the thesis of new state capitalism. Remunicipalisation refers to a process whereby towns, cities and sub-national regions take previously privatised services and infrastructures back into public ownership. Remunicipalisation has led to the emergence of regionally- and municipally-owned state enterprises across a wide range of sectors including water, energy, waste, transport, education, (tele)communications, and health and social care. Engaging with the nature of the ‘new’ state capitalism, and particularly challenging its theoretically restrictive understanding of the state as ‘market enabler’, we highlight that remunicipalisations have often emerged in response to the failed promise of neoliberal capitalism to improve the quality and efficiency of public services through the (supposed) competitiveness of the free market. Like neoliberalism, remunicipalisations take spatially diverse and variegated forms as market-driven logics interact with other political and economic determinations. As such, remunicipalisations often encompass critiques of neoliberal governance and market volatility, and instead focus on the potential of regional wealth creation as well as stabilising local market dynamics through diversifying ownership forms. Drawing upon our ongoing empirical work on German remunicipalisation, we aim to foreground how multiple determinations at work in the German political economy at different spatial scales shape its particular trajectory out of neoliberal mutation. We show how the remunicipalisation of energy provider TEAG has enabled the local state to intervene and diversify the uneven economic geographies in the state of Thuringia.
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Ventura-Espejo, Laura, Inés Gracia-Darder, Silvia Escribá-Bori, Eva Regina Amador-González, Ana Martín-Santiago i Jan Ramakers. "Patient with H syndrome, cardiogenic shock, multiorgan infiltration, and digital ischemia". Pediatric Rheumatology 19, nr 1 (30.06.2021). http://dx.doi.org/10.1186/s12969-021-00586-2.
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Abstract Background H syndrome (HS) is a rare autoinflammatory disease caused by a mutation in the solute carrier family 29, member 3 (SCL29A3) gene. It has a variable clinical presentation and little phenotype-genotype correlation. The pathognomonic sign of HS is cutaneous hyperpigmentation located mainly in the inner thighs and often accompanied by other systemic manifestations. Improvement after tocilizumab treatment has been reported in a few patients with HS. We report the first patient with HS who presented cardiogenic shock, multiorgan infiltration, and digital ischemia. Case presentation 8-year-old boy born to consanguineous parents of Moroccan origin who was admitted to the intensive care unit during the Coronavirus Disease-2019 (COVID-19) pandemic with tachypnoea, tachycardia, and oliguria. Echocardiography showed dilated cardiomyopathy and severe systolic dysfunction compatible with cardiogenic shock. Additionally, he presented with multiple organ dysfunction syndrome. SARS-CoV-2 polymerase chain reaction (PCR) and antibody detection by chromatographic immunoassay were negative. A previously ordered gene panel for pre-existing sensorineural hearing loss showed a pathological mutation in the SCL29A3 gene compatible with H syndrome. Computed tomography scan revealed extensive alveolar infiltrates in the lungs and multiple poor defined hypodense lesions in liver, spleen, and kidneys; adenopathy; and cardiomegaly with left ventricle subendocardial nodules. Invasive mechanical ventilation, broad antibiotic and antifungal coverage showed no significant response. Therefore, Tocilizumab as compassionate use together with pulsed intravenous methylprednisolone was initiated. Improvement was impressive leading to normalization of inflammation markers, liver and kidney function, and stabilising heart function. Two weeks later, he was discharged and has been clinically well since then on two weekly administration of Tocilizumab. Conclusions We report the most severe disease course produced by HS described so far in the literature. Our patient’s manifestations included uncommon, new complications such as acute heart failure with severe systolic dysfunction, multi-organ cell infiltrate, and digital ischemia. Most of the clinical symptoms of our patient could have been explained by SARS-CoV-2, demonstrating the importance of a detailed differential diagnosis to ensure optimal treatment. Although the mechanism of autoinflammation of HS remains uncertain, the good response of our patient to Tocilizumab makes a case for the important role of IL-6 in this syndrome and for considering Tocilizumab as a first-line treatment, at least in severely affected patients.