RESUMO
Kidney- and brain-expressed protein (KIBRA), a multifunctional scaffold protein with around 20 known binding partners, is involved in memory and cognition, organ size control via the Hippo pathway, cell polarity, and membrane trafficking. KIBRA includes tandem N-terminal WW domains, a C2 domain, and motifs for binding atypical PKC and PDZ domains. A naturally occurring human KIBRA variant involving residue changes at positions 734 (Met-to-Ile) and 735 (Ser-to-Ala) within the C2 domain affects cognitive performance. We have elucidated 3D structures and calcium- and phosphoinositide-binding properties of human KIBRA C2 domain. Both WT and variant C2 adopt a canonical type I topology C2 domain fold. Neither Ca2+ nor any other metal ion was bound to WT or variant KIBRA C2 in crystal structures, and Ca2+ titration produced no significant reproducible changes in NMR spectra. NMR and X-ray diffraction data indicate that KIBRA C2 binds phosphoinositides via an atypical site involving ß-strands 5, 2, 1, and 8. Molecular dynamics simulations indicate that KIBRA C2 interacts with membranes via primary and secondary sites on the same domain face as the experimentally identified phosphoinositide-binding site. Our results indicate that KIBRA C2 domain association with membranes is calcium-independent and involves distinctive C2 domain-membrane relative orientations.
Assuntos
Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Domínios C2 , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação ProteicaRESUMO
Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.
Assuntos
Proteínas de Bactérias/metabolismo , Plaquetas/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/metabolismo , Selectina-P/metabolismo , Staphylococcus aureus/metabolismo , Plaquetas/patologia , Proteínas Sanguíneas/metabolismo , Humanos , Monócitos/patologiaRESUMO
Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.
Assuntos
Proteínas Arqueais/metabolismo , Peptídeo Sintases/metabolismo , Processamento de Proteína Pós-Traducional , Thermoplasma/enzimologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Sítios de Ligação , Cristalografia por Raios X , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/química , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/genética , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoilação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácido Tióctico/química , Ácido Tióctico/metabolismoRESUMO
As part of the Biomedical Sciences undergraduate degree course students are required to apply biological principles to the interpretation of clinical case studies and the diagnosis of patients. Case study-based learning, i.e., application of knowledge to patient diagnosis, is new to most students as case studies do not form part of non-applied A level courses in biological sciences. This approach is an example of Problem Based Learning (PBL) which has been shown to support higher levels of student learning, encouraging critical thinking and analysis. PBL approaches have also been shown to increase academic satisfaction and student engagement. In recent years we have observed a downwards trend in student engagement and historically student performance in applied case study-based assessments to be lower than that observed for assessments based on detailing fundamental biological principles. We hypothesised that PBL teaching delivery would support students in preparing for case study-based assessments, helping them to demonstrate their critical evaluation and problem-solving skills, and hence, improve student performance. We also hypothesised that the student learning experience would be enhanced by a PBL teaching delivery approach which would improve overall engagement. We therefore redesigned a second year Biomedical Sciences degree haematology and clinical biochemistry unit: "Blood Science," with a stronger focus on PBL, including case study focussed activities throughout the unit. We subsequently analysed whether this PBL-focussed unit design improved student experience and feedback, student engagement and student confidence for biomedical science undergraduate students. We present here, our teaching strategy and the impact our changes had on student feedback for the 21/22 and 22/23 academic years. Our findings demonstrate that case study-based activities and tutorial PBL exercises, when incorporated into the curriculum design, can improve student experience in the Biomedical Sciences and other biological science undergraduate degree courses.
Assuntos
Currículo , Aprendizagem Baseada em Problemas , Humanos , EstudantesRESUMO
Multimerin-1 (MMRN1) is a platelet protein with a role in haemostasis and coagulation. It is also present in endothelial cells (ECs) and the extracellular matrix (ECM), where it may be involved in cell adhesion, but its molecular functions and protein-protein interactions in these cellular locations have not been studied in detail yet. In recent years, MMRN1 has been identified as a differentially expressed gene (DEG) in various cancers and it has been proposed as a possible cancer biomarker. Some evidence suggest that MMRN1 expression is regulated by methylation, protein interactions, and non-coding RNAs (ncRNAs) in different cancers. This raises the questions if a functional role of MMRN1 is being targeted during cancer development, and if MMRN1's differential expression pattern correlates with cancer progression. As a result, it is timely to review the current state of what is known about MMRN1 to help inform future research into MMRN1's molecular mechanisms in cancer.
Assuntos
Células Endoteliais , Neoplasias , Plaquetas/metabolismo , Proteínas Sanguíneas/genética , Células Endoteliais/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMO
We describe the use of HTML5P (H5P) content collaboration framework to deliver an interactive, online alternative to an assessed laboratory practical on the Biomedical Cell Biology unit at the Manchester Metropolitan University, U.K. H5P is free, open-source technology to deliver bespoke interactive, self-paced online sessions. To determine if the use of H5P affected learning and student attainment, we compared the student grades among three cohorts: the 18/19 cohort who had 'wet' laboratory classes, the 19/20 cohort who had 'wet' laboratory classes with additional video support and the 20/21 cohort who had the H5P alternative. Our analysis shows that students using the H5P were not at a disadvantage to students who had 'wet' laboratory classes with regard to assessment outcomes. Student feedback, mean grade attained and an upward trend in the number of students achieving first-class marks (≥70%), indicate H5P may enhance students' learning experience and be a valuable learning source augmenting traditional practical classes in the future.
Assuntos
Aprendizagem , HumanosRESUMO
The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.
Assuntos
Proteínas Arqueais/metabolismo , Cetona Oxirredutases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Recombinantes/metabolismo , Thermoplasma/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cromatografia em Gel , Clonagem Molecular , Biologia Computacional , Cetona Oxirredutases/química , Cetona Oxirredutases/genética , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Developing generic strategies for building adaptable or multifunctional bioplatforms is challenging, in particular because protein immobilization onto surfaces often causes loss of protein function and because multifunctionality usually necessitates specific combinations of heterogeneous elements. Here, we introduce a generic, modular bioplatform construction strategy that uses cage-like supramolecular multienzyme complexes as highly adaptable building blocks immobilized directly and noncovalently on graphene. Thermoplasma acidophilum dihydrolipoyl acyltransferase (E2) supramolecular complexes organize as a monolayer or can be controllably transferred onto graphene, preserving their supramolecular form with specific molecular recognition capability and capacity for engineering multifunctionality. This E2-graphene platform can bind enzymes (here, E1, E2's physiological partner) without loss of enzyme function; in this test case, E1 catalytic activity was detected on E2-graphene over 6 orders of magnitude in substrate concentration. The E2-graphene platform can be multiplexed via patterned cotransfer of differently modified E2 complexes. As the E2 complexes are robust and highly customizable, E2-graphene is a platform onto which multiple functionalities can be built.
Assuntos
Grafite/química , Complexos Multienzimáticos , Proteínas , ThermoplasmaRESUMO
Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.
RESUMO
Transient transfection of human embryonic kidney cells (HEK 293) enables the rapid and affordable lab-scale production of recombinant proteins. In this chapter protocols for the expression and purification of both secreted and intracellular proteins using transient expression in HEK 293 cells are described.
Assuntos
Escherichia coli/genética , Células HEK293/metabolismo , Mamíferos/genética , Proteínas Recombinantes/genética , Animais , Escherichia coli/metabolismo , Humanos , Mamíferos/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção/métodosRESUMO
Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea, the extension of comparative genomic pathway profiling to broader metabolic and homeostasis networks should be useful in revealing characteristics from metagenomic datasets related to adaptations to diverse environments.
Assuntos
Oxirredutases do Álcool/genética , Archaea/enzimologia , Archaea/genética , Genômica/métodos , Lipoilação , Família Multigênica , Aerobiose , Oxirredutases do Álcool/metabolismo , Anaerobiose , Biotina/metabolismo , Genes Arqueais , Ligases/genética , Filogenia , Especificidade por Substrato , Ácido Tióctico/metabolismoRESUMO
Members of the 2-oxoacid dehydrogenase multienzyme complex family play a key role in the pathways of central metabolism. Post-translational lipoylation of the dihydrolipoyl acyltransferase component of these complexes is essential for their activity, the lipoyllysine moiety performing the transfer of substrates and intermediates between the different active sites within these multienzyme systems. We have previously shown that the thermophilic archaeon, Thermoplasma acidophilum, has a four-gene cluster encoding the components of such a complex, which, when recombinantly expressed in Escherichia coli, can be assembled into an active multienzyme in vitro. Crucially, the E. coli host carries out the required lipoylation of the archaeal dihydrolipoyl acyltransferase component. Because active 2-oxoacid dehydrogenase multienzyme complexes have never been detected in any archaeon, the question arises as to whether Archaea possess a functional lipoylation system. In this study, we report the cloning and heterologous expression of two genes from Tp. acidophilum whose protein products together show significant sequence identity with the single lipoate protein ligase enzyme of bacteria. We demonstrate that both recombinantly expressed Tp. acidophilum proteins are required for lipoylation of the acyltransferase, and that the two proteins associate together to carry out this post-translational modification. From the published DNA sequences, we suggest the presence of functional transcriptional and translational regulatory elements, and furthermore we present preliminary evidence that lipoylation occurs in vivo in Tp. acidophilum. This is the first report of the lipoylation machinery in the Archaea, which is unique in that the catalytic activity is dependent on two separate gene products.