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1.
Proc Natl Acad Sci U S A ; 119(10): e2119529119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35238631

RESUMO

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.


Assuntos
Homeostase , Neurônios/metabolismo , Conformação de Ácido Nucleico , RNA de Transferência/metabolismo , Animais , Pareamento de Bases , Córtex Cerebral/enzimologia , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Mutação Puntual , Processamento de Proteína Pós-Traducional , RNA de Transferência/química , RNA de Transferência/genética , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismo , Especificidade por Substrato
2.
Electrophoresis ; 45(17-18): 1597-1605, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38577828

RESUMO

Capillary electrophoresis (CE) interfaced to mass spectrometry (MS) with electrospray ionization typically incorporates acidic additives or organic solvents to assist in ionization. Vibrating sharp-edge spray ionization (VSSI) is a voltage-free method to interface CE and MS that does not require these additives, making it appealing for protein analyses. CE-VSSI nanoflow sheath separations are performed with low ionic strength aqueous solutions in the sheath to reduce suppression. Serine is also included in the sheath to reduce analyte adduction. Proteins are detected in the 2.5-10 µM range, corresponding to an injected mass range of 0.1-1.2 ng. The anionic proteins ß-lactoglobulin and transferrin are resolved using an unmodified fused silica capillary because they do not exhibit nonspecific surface adsorption. Conversely, separations of cationic proteins cytochrome c, ribonuclease A, and α-chymotrypsinogen A in an unmodified capillary require acidic background electrolytes to overcome adsorption. Alternatively, a semipermanent coating comprised self-assembled lipids overcomes surface adsorption at a neutral pH. Separations with zwitterionic and hybrid cationic coatings are complete within 15 or 6 min, respectively. The dimeric form of triosephosphate isomerase was observed at a 60 µM, corresponding to a mass of 19 ng, by dropping the temperature of the MS inlet.


Assuntos
Eletroforese Capilar , Proteínas , Espectrometria de Massas por Ionização por Electrospray , Eletroforese Capilar/métodos , Proteínas/análise , Proteínas/química , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais
3.
Nucleic Acids Res ; 49(16): 9444-9458, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387688

RESUMO

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.


Assuntos
Archaea/enzimologia , Magnésio/metabolismo , RNA Arqueal/genética , Ribonuclease P/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Precursores de RNA/genética , RNA Arqueal/ultraestrutura , RNA Catalítico , Ribonuclease P/ultraestrutura
4.
Biochemistry ; 60(24): 1876-1884, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34100589

RESUMO

Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein-specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography-mass spectrometry-based method for direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC-OBE-nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.


Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Proteínas/isolamento & purificação , Cromatografia Líquida/métodos , Histidina/química , Estudo de Prova de Conceito
5.
Nucleic Acids Res ; 45(12): 7432-7440, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28525600

RESUMO

RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented 'double K-turn' module in type A and type M archaeal RPR variants.


Assuntos
Proteínas Arqueais/química , Regulação da Expressão Gênica em Archaea , Methanocaldococcus/enzimologia , Pyrococcus furiosus/enzimologia , RNA Arqueal/química , RNA de Transferência/química , Ribonuclease P/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Methanocaldococcus/genética , Mathanococcus/enzimologia , Mathanococcus/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Pyrococcus furiosus/genética , Precursores de RNA , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo
6.
Nucleic Acids Res ; 42(21): 13328-38, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25361963

RESUMO

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg(2+)-dependent 5' maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe-modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae-EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.


Assuntos
Proteínas Arqueais/química , Pyrococcus furiosus , RNA Arqueal/química , Proteínas de Ligação a RNA/química , Ribonuclease P/química , Substituição de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Pyrococcus furiosus/genética , RNA Arqueal/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease P/metabolismo
7.
Angew Chem Int Ed Engl ; 53(43): 11483-7, 2014 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-25195671

RESUMO

We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.


Assuntos
Espectrometria de Massas/métodos , Pyrococcus furiosus/enzimologia , Ribonuclease P/metabolismo , Catálise , Ribonuclease P/química
8.
bioRxiv ; 2023 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-37066191

RESUMO

Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign problems, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 15 new homo-oligomers and recombined them to make 17 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system share a common backbone, and hence are ideal building blocks for generating and functionalizing larger symmetric assemblies.

9.
Mol Genet Genomics ; 286(5-6): 359-69, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21987179

RESUMO

RNase P catalyzes 5'-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter's presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5'-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.


Assuntos
Proteínas de Bactérias , Evolução Biológica , Eucariotos/enzimologia , Ribonuclease P/fisiologia , Eucariotos/genética , Transferência Genética Horizontal , Subunidades Proteicas , Homologia de Sequência de Aminoácidos
10.
Methods Mol Biol ; 2167: 147-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712919

RESUMO

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.


Assuntos
Proteínas Arqueais/química , Pegada de DNA/métodos , Ácido Edético/análogos & derivados , Radical Hidroxila/química , Dobramento de RNA/genética , RNA/química , Ribonuclease P/metabolismo , Sítios de Ligação , Ácido Edético/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Reversa , Ribonuclease P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA
11.
Methods Mol Biol ; 2167: C1, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33044739

RESUMO

Owing to an oversight, the words "Reverse" and "Cleaved" in Subheading 2.3.3 of Chapter 9 were spelt incorrectly in the book.

12.
Methods Enzymol ; 659: 37-70, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34752295

RESUMO

Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is routinely used to screen a variety of overexpression conditions (e.g., host, medium, inducer concentration, post-induction temperature and/or incubation time) and to assess the purity of the final product, its limitations, including aberrant protein migration due to compositional eccentricities or incomplete denaturation, often preclude firm conclusions regarding the extent of overexpression and/or purification. Therefore, we recently reported an automated liquid chromatography-mass spectrometry-based strategy that couples immobilized metal affinity chromatography (IMAC) with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS) to directly analyze cell lysates for the presence of target proteins. IMAC-OBE-nMS can be used to assess whether target proteins (1) are overexpressed in soluble form, (2) bind and elute from an IMAC resin, (3) oligomerize, and (4) have the expected mass. Here, we use four poly-His-tagged proteins to demonstrate the potential of IMAC-OBE-nMS for expedient optimization of overexpression and purification conditions for recombinant protein production.


Assuntos
Histidina , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Histidina/metabolismo , Espectrometria de Massas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
13.
Methods Enzymol ; 659: 71-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34752299

RESUMO

The ubiquitous ribonucleoprotein (RNP) form of RNase P catalyzes the Mg2+-dependent cleavage of the 5' leader of precursor-transfer RNAs. The rate and fidelity of the single catalytic RNA subunit in the RNase P RNP is significantly enhanced by association with protein cofactors. While the bacterial RNP exhibits robust activity at near-physiological Mg2+ concentrations with a single essential protein cofactor, archaeal and eukaryotic RNase P are dependent on up to 5 and 10 protein subunits, respectively. Archaeal RNase P-whose proteins share eukaryotic homologs-is an experimentally tractable model for dissecting in a large RNP the roles of multiple proteins that aid an RNA catalyst. We describe protocols to assemble RNase P from Methanococcus maripaludis, a methanogenic archaeon. We present strategies for tag-less purification of four of the five proteins (the tag from the fifth is removed post-purification), an approach that helps reconstitute the RNase P RNP with near-native constituents. We demonstrate the value of native mass spectrometry (MS) in establishing the accurate masses (including native oligomers and modifications) of all six subunits in M. maripaludis RNase P, and the merits of mass photometry (MP) as a complement to native MS for characterizing the oligomeric state of protein complexes. We showcase the value of native MS and MP in revealing time-dependent modifications (e.g., oxidation) and aggregation of protein subunits, thereby providing insights into the decreased function of RNase P assembled with aged preparations of recombinant subunits. Our protocols and cautionary findings are applicable to studies of other cellular RNPs.


Assuntos
Proteínas Arqueais , RNA Catalítico , Archaea , Proteínas Arqueais/metabolismo , RNA , Precursores de RNA , RNA Catalítico/metabolismo , Ribonuclease P/química , Ribonuclease P/genética , Ribonuclease P/metabolismo
14.
J Proteomics ; 249: 104360, 2021 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-34481086

RESUMO

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SIGNIFICANCE: Genetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.


Assuntos
Peptídeos , Proteômica , Medicina Legal , Humanos , Espectrometria de Massas , Proteoma , Tripsina
15.
Sci Rep ; 8(1): 3379, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29467394

RESUMO

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP+ to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.


Assuntos
Bioensaio/métodos , Butanóis/metabolismo , Clostridium/metabolismo , Saccharomyces cerevisiae/metabolismo , Álcool Desidrogenase/metabolismo , Biocombustíveis/microbiologia , Meios de Cultura/metabolismo , Etanol/metabolismo , Fermentação/fisiologia , NADP/metabolismo
16.
J Clin Med Res ; 9(8): 709-718, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28725320

RESUMO

BACKGROUND: Approximately 75% of clinically definite long QT syndrome (LQTS) cases are caused by mutations in the KCNQ1, KCNH2 and SCN5A genes. Of these mutations, a small proportion (3.2-9.2%) are predicted to affect splicing. These mutations present a particular challenge in ascribing pathogenicity. METHODS: Here we report an analysis of the transcriptional consequences of two mutations, one in the KCNQ1 gene (c.781_782delinsTC) and one in the SCN5A gene (c.2437-5C>A), which are predicted to affect splicing. We isolated RNA from lymphocytes and used a directed PCR amplification strategy of cDNA to show mis-spliced transcripts in mutation-positive patients. RESULTS: The loss of an exon in each mis-spliced transcript had no deduced effect on the translational reading frame. The clinical phenotype corresponded closely with genotypic status in family members carrying the KCNQ1 splice variant, but not in family members with the SCN5A splice variant. These results are put in the context of a literature review, where only 20% of all splice variants reported in the KCNQ1, KCNH2 and SCN5A gene entries in the HGMDPro 2015.4 database have been evaluated using transcriptional assays. CONCLUSIONS: Prediction programmes play a strong role in most diagnostic laboratories in classifying variants located at splice sites; however, transcriptional analysis should be considered critical to confirm mis-splicing. Critically, this study shows that genuine mis- splicing may not always imply clinical significance, and genotype/phenotype cosegregation remains important even when mis-splicing is confirmed.

17.
Biomolecules ; 6(2)2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-27104580

RESUMO

RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5'-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs.


Assuntos
Archaea/metabolismo , Proteínas Arqueais/metabolismo , Ribonuclease P/metabolismo , Proteínas Arqueais/química , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonuclease P/química
18.
Sultan Qaboos Univ Med J ; 15(2): e218-25, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26052455

RESUMO

OBJECTIVES: Missense variants are very commonly detected when screening for mutations in the BRCA1 and BRCA2 genes. Pathogenic mutations in the BRCA1 and BRCA2 genes lead to an increased risk of developing breast, ovarian, prostate and/or pancreatic cancer. This study aimed to assess the predictive capability of in silico programmes and mutation databases in assisting diagnostic laboratories to determine the pathogenicity of sequence-detectable mutations. METHODS: Between July 2011 and April 2013, an analysis was undertaken of 13 missense BRCA gene variants that had been detected in patients referred to the Genetic Health Services New Zealand (Northern Hub) for BRCA gene analysis. The analysis involved the use of 13 in silico protein prediction programmes, two in silico transcript analysis programmes and the examination of three BRCA gene databases. RESULTS: In most of the variants, the analysis showed different in silico interpretations. This illustrates the interpretation challenges faced by diagnostic laboratories. CONCLUSION: Unfortunately, when using online mutation databases and carrying out in silico analyses, there is significant discordance in the classification of some missense variants in the BRCA genes. This discordance leads to complexities in interpreting and reporting these variants in a clinical context. The authors have developed a simple procedure for analysing variants; however, those of unknown significance largely remain unknown. As a consequence, the clinical value of some reports may be negligible.

19.
Sultan Qaboos Univ Med J ; 15(1): e58-70, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25685387

RESUMO

OBJECTIVES: Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, massively parallel sequencing (MPS) can accommodate increasing test demands and financial constraints. This study aimed to establish a simple workflow to amplify and screen all coding regions of the BRCA1 and BRCA2 (BRCA1/2) genes by Sanger-based sequencing as well as to assess a MPS approach encompassing multiplex polymerase chain reaction (PCR) and pyrosequencing. METHODS: This study was conducted between July 2011 and April 2013. A total of 20 patients were included in the study who had been referred to Genetic Health Services New Zealand (Northern Hub) for BRCA1/2 mutation screening. Patients were randomly divided into a MPS evaluation and validation cohort (n = 10 patients each). Primers were designed to amplify all coding exons of BRCA1/2 (28 and 42 primer pairs, respectively). Primers overlying known variants were avoided to circumvent allelic drop-out. The MPS approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were filtered on the basis of their frequency and sequence depth. RESULTS: Sanger-based sequencing of PCR-amplified coding regions was successfully achieved. Sensitivity and specificity of the combined MPS/homopolymer protocol was determined to be 100% and 99.5%, respectively. CONCLUSION: In comparison to traditional Sanger-based sequencing, the MPS workflow led to a reduction in both cost and analysis time for BRCA1/2 screening. MPS analysis achieved high analytical sensitivity and specificity, but required complementary fragment analysis combined with Sanger-based sequencing confirmation in some instances.

20.
Simul Healthc ; 10(3): 146-53, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25844702

RESUMO

INTRODUCTION: Patient safety during emergency department procedural sedation (EDPS) can be difficult to study. Investigators sought to delineate and experimentally assess EDPS performance and safety practices of senior-level emergency medicine residents through in situ simulation. METHODS: Study sessions used 2 pilot-tested EDPS scenarios with critical action checklists, institutional forms, embedded probes, and situational awareness questionnaires. An experimental informatics system was separately developed for bedside EDPS process guidance. Postgraduate year 3 and 4 subjects completed both scenarios in randomized order; only experimental subjects were provided with the experimental system during second scenarios. RESULTS: Twenty-four residents were recruited into a control group (n = 12; 6.2 ± 7.4 live EDPS experience) and experimental group (n = 12; 11.3 ± 8.2 live EDPS experience [P = 0.10]). Critical actions for EDPS medication selection, induction, and adverse event recognition with resuscitation were correctly performed by most subjects. Presedation evaluations, sedation rescue preparation, equipment checks, time-outs, and documentation were frequently missed. Time-outs and postsedation assessments increased during second scenarios in the experimental group. Emergency department procedural sedation safety probe detection did not change across scenarios in either group. Situational awareness scores were 51% ± 7% for control group and 58% ± 12% for experimental group. Subjects using the experimental system completed more time-outs and scored higher Simulation EDPS Safety Composite Scores, although without comprehensive improvements in EDPS practice or safety. CONCLUSIONS: Study simulations delineated EDPS and assessed safety behaviors in senior emergency medicine residents, who exhibited the requisite medical knowledge base and procedural skill set but lacked some nontechnical skills that pertain to emergency department microsystem functions and patient safety. The experimental system exhibited limited impact only on in-simulation time-out compliance.


Assuntos
Serviço Hospitalar de Emergência/organização & administração , Hipnóticos e Sedativos/administração & dosagem , Internato e Residência/organização & administração , Ressuscitação/métodos , Treinamento por Simulação/organização & administração , Adulto , Lista de Checagem , Tomada de Decisões , Feminino , Humanos , Masculino , Segurança do Paciente
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