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1.
Artigo em Inglês | MEDLINE | ID: mdl-38958725

RESUMO

PURPOSE: Fresh-frozen specimen availability and cost may be a barrier for initiation of biomechanical studies where soft tissue is used in a construct with other medical devices. The impact of soft tissue preservation method on the outcomes of biomechanical studies in the specific case of graft-suture constructs is relatively unexplored. This study aimed to observe peak loads and failure modes in biomechanical testing of fresh-frozen (FF) versus formalin embalmed (FE) quadriceps tendon (QT) graft-suture constructs for soft tissue fixation in ACLR and assess suitability of FE QT graft constructs for load-to-fail testing. METHODS: Twenty QT grafts were harvested from human cadaver specimens. Ten grafts came from fresh-frozen donors and 10 from embalmed donors. All grafts were prepared with the modified Prusik knot using a braided composite suture and subjected to tensile loading. Comparisons between the biomechanical properties of the graft-suture constructs were made with unpaired t tests with α = 0.05. RESULTS: FE and FF constructs displayed similar peak loads and failure modes. FF constructs had greater elongation after pre-tensioning than FE (7.3 vs. 5.5 mm, p = 0.02) and greater elongation after cyclic loading than FE constructs (17.5 vs. 10.5 mm, p = 0.01). Hysteresis was greater for FF constructs at the 50th, 100th, 150th, and 200th cycle (p = 0.02, p = 0.07, p < 0.001, p = 0.004, respectively). FE constructs were stiffer than fresh-frozen (103 vs. 84 N/mm, p < 0.001). CONCLUSION: FE constructs were significantly stiffer but displayed similar peak load and failure mode to FF which was reflective of the strength of the suture material. FE grafts can offer an alternative to FF grafts in graft-suture constructs for biomechanical studies where load at failure and knot security and strength is of main interest.

2.
BMJ Open ; 13(5): e070920, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37253502

RESUMO

INTRODUCTION: Complex regional pain syndrome (CRPS) is a disabling and distressing chronic pain condition characterised by a range of sensory, motor, autonomic and trophic symptoms. UK guidelines recommend therapy interventions to help normalise touch perception through self-administered tactile and thermal desensitisation activities. Interventions have been developed, aiming to help individuals broaden their sensory experience, thereby relieving chronic pain. However, therapy-led interventions often experience practical constraints and poor adherence. In response, a sensory training system (STS) device has been designed for unsupervised independent home-use. METHODS: This proof-of-concept study aims to explore whether people with CRPS use the device at home for 30 minutes a day for 30 days. Secondary aims are to determine whether the STS device will change tactile acuity and perceived levels of pain intensity, pain interference, sensitivity or feelings towards the affected limb. We will seek to recruit 20 eligible participants. Participants will be asked to measure tactile acuity using a two-point discrimination assessment, complete an online questionnaire before and after use of the device and complete a daily diary. On completion of the 30-day use, participants will be invited to take part in a semi-structured interview to explore their experiences of using the device. ANALYSIS: Pain intensity and pain interference will be scored using the online Assessment Center Scoring Service or using the look-up table in the PROMIS scoring manual. The remaining questionnaire data, including tactile acuity results, and device-use data, including frequency and duration of use, will be analysed using descriptive statistics. Qualitative data will be thematically analysed. ETHICS AND DISSEMINATION: London-Stanmore Research Ethics Committee provided a favourable opinion on 19 April 2021 (ref 21/LO/0200). The NHS Health Research Authority, UK, approved this study on 7 June 2021. Dissemination will include peer-reviewed publications, presentations at conferences, social media and reports to the funder and patient charities. TRIAL REGISTRATION NUMBER: ISRCTN89099843.


Assuntos
Dor Crônica , Síndromes da Dor Regional Complexa , Humanos , Dor Crônica/terapia , Síndromes da Dor Regional Complexa/terapia , Tato , Medição da Dor/métodos , Inquéritos e Questionários
3.
Eur J Orthop Surg Traumatol ; 33(4): 1067-1074, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-35362777

RESUMO

PURPOSE: The three most commonly used autografts for anterior cruciate ligament reconstruction (ACL) are: bone-patellar tendon-bone (BTB), hamstring tendons (HT), and quadriceps tendon (QT). A cadaveric study was performed to determine if there were any differences in mechanical and structural properties under biomechanical testing. METHODS: Twenty-seven graft specimens were harvested from 9 human cadaveric legs. Mean donor age was 75.2 years (range 53-85 years). Twenty-two specimens (8 HT, 7 QT, and 7 BTB) completed cyclic preconditioning from 50 to 800 N for 200 cycles and a load to failure test at an extension rate of 1 mm/s. Structural and mechanical properties of BTB, HT, and QT grafts were compared using a one-way ANOVA and Tukey's honest significant difference. RESULTS: There was no difference in the ultimate load to failure (N) across all 3 graft types (p = 0.951). Quadriceps tendon demonstrated greater cross-sectional area (mm2) when compared to both HT and BTB (p = 0.001) and was significantly stiffer (N/mm) than HT but not BTB (p = 0.004). Stress (N/mm2) of the HT at ultimate load was greater than QT but not BTB (p = 0.036). Elastic modulus (MPa) of HT was greater than both QT and BTB (p = 0.016). CONCLUSION: There was no difference in the ultimate load to failure of BTB, HT, and QT grafts harvested from the same specimens. All 3 grafts had similar loads to failure with a significant increase in stiffness when compared to the native ACL. Furthermore, QT demonstrated more favourable structural properties compared to HT and BTB with greater cross-sectional area to both HT and BTB and greater stiffness compared to HT.


Assuntos
Lesões do Ligamento Cruzado Anterior , Reconstrução do Ligamento Cruzado Anterior , Tendões dos Músculos Isquiotibiais , Humanos , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Tendões dos Músculos Isquiotibiais/transplante , Enxertos Osso-Tendão Patelar-Osso/cirurgia , Fenômenos Biomecânicos , Tendões/cirurgia , Transplante Autólogo , Cadáver , Lesões do Ligamento Cruzado Anterior/cirurgia
4.
Clin Orthop Relat Res ; 480(7): 1403-1410, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35238847

RESUMO

BACKGROUND: ACL graft-suture fixation can be constructed with needle or needleless techniques. Needleless techniques have the advantage of decreased injury, preparation time, and cost. The Nice knot is common among upper extremity procedures, and has been shown to have higher load to failure and less elongation compared with other double loop knots; however, there are no studies that have looked at its use for ACL graft-suture construct to determine whether it offers less elongation relative to other needleless techniques. QUESTIONS/PURPOSES: In a cadaver quadriceps tendon model, we asked: (1) Does the Nice knot have less elongation than the Prusik knot? (2) Does the Nice knot have increased peak load and stiffness compared with the Prusik knot? (3) What were the modes of failure of each knot? METHODS: Sixteen quadriceps tendon grafts were harvested from 16 cadaver knee specimens. The median (range) age of the donors was 80 years (70 to 96) and included three male and five female donors. Eight grafts were prepared with the Prusik knot and eight with the Nice knot using a braided polyblend suture. The graft-suture constructs were mounted in a materials testing machine and subjected to a tensile loading protocol beginning with pretensioning of three cycles from 0 to 100 N at 1 Hz followed by a constant load of 50 N for 1 minute then cyclic loading of 200 cycles from 50 to 200 N at 1 Hz. The constructs were loaded to failure as the final step of the loading protocol. Elongations of the construct after each loading step, peak load, stiffness, and graft cross-sectional area were compared. RESULTS: Construct elongations (median [IQR]) for the Nice knot were lower than that of the Prusik knot after pretensioning (4.4 mm [0.8] versus 5.7 mm [1.4]; p = 0.02), preloading (0.6 mm [0.3] versus 1.0 mm [0.3]; p = 0.005), and cyclic loading (7.4 mm [1.4] versus 10.9 mm [2.1]; p = 0.005). Peak load was not different for the Prusik knot construct compared with the Nice knot (334 N [43] versus 312 N [13]; p = 0.08). Stiffness of the Prusik knot construct (103 N/mm [17]) was no different than the Nice knot construct (110 N/mm [13]; p = 0.13). Graft cross-sectional area of the Prusik knot constructs (85 mm2 [35]) were similar to the grafts of the Nice knot constructs (97 mm2 [31]; p = 0.28). Failure mode of the constructs did not differ between groups; it was caused by suture rupture near the knots that secured the free suture ends to the machine and was seen in all 16 tests. CONCLUSIONS: The results of this biomechanical study show that the Nice knot construct has similar or greater biomechanical properties compared with the Prusik knot in the graft suture construct, although the magnitude of the differences are not likely to the level of clinical importance. CLINICAL RELEVANCE: The Nice knot offers an attractive alternative option for needleless ACL graft preparation technique. Future studies should consider comparison to established needle techniques such as Krackow or whipstitch and testing in an intraarticular component in an in vivo model.


Assuntos
Técnicas de Sutura , Tendões , Idoso de 80 Anos ou mais , Fenômenos Biomecânicos , Cadáver , Feminino , Humanos , Masculino , Suturas , Tendões/cirurgia
5.
Angew Chem Int Ed Engl ; 60(9): 4689-4697, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33320993

RESUMO

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.


Assuntos
Complexo I de Transporte de Elétrons/química , Flavina-Adenina Dinucleotídeo/metabolismo , Mitocôndrias/metabolismo , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Flavina-Adenina Dinucleotídeo/química , Humanos , Fosforilação Oxidativa , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
6.
J Rheumatol ; 48(6): 867-876, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33132218

RESUMO

OBJECTIVE: The modified Rodnan skin score (mRSS) remains the preferred method for skin assessment in systemic sclerosis (SSc). There are concerns regarding high interobserver variability of mRSS and negative clinical trials utilizing mRSS as the primary endpoint. High-frequency ultrasound (HFUS) allows objective assessment of cutaneous fibrosis in SSc. We investigated the relationship between HFUS with both mRSS and dermal collagen. METHODS: Skin thickness (ST), echogenicity, and novel shear wave elastography (SWE) were assessed in 53 patients with SSc and 15 healthy controls (HCs) at the finger, hand, forearm, and abdomen. The relationship between HFUS parameters with mRSS (n = 53) and dermal collagen (10 patients with SSc and 10 HCs) was investigated. Intraobserver repeatability of HFUS was calculated using intraclass correlation coefficients (ICCs). RESULTS: HFUS assessment of ST (hand/forearm) and SWE (finger/hand) correlated with local mRSS at some sites. Subclinical abnormalities in ST, echogenicity, and SWE were present in clinically uninvolved SSc skin. Additionally, changes in echogenicity and SWE were sometimes apparent despite objectively normal ST on HFUS. ST, SWE, and local mRSS correlated strongly with collagen quantification (r = 0.697, 0.709, 0.649, respectively). Intraobserver repeatability was high for all HFUS parameters (ICCs for ST = 0.946-0.978; echogenicity = 0.648-0.865; and SWE = 0.953-0.973). CONCLUSION: Our data demonstrate excellent reproducibility and reassuring convergent validity with dermal collagen content. Detection of subclinical abnormalities is an additional benefit of HFUS. The observed correlations with collagen quantification support further investigation of HFUS as an alternative to mRSS in clinical trial settings.


Assuntos
Escleroderma Sistêmico , Colágeno , Mãos/diagnóstico por imagem , Humanos , Reprodutibilidade dos Testes , Escleroderma Sistêmico/diagnóstico por imagem , Pele/diagnóstico por imagem , Ultrassonografia
7.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , Proteínas de Ligação a RNA/ultraestrutura , RNA Polimerase Dependente de RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
8.
J Acoust Soc Am ; 144(5): 3036, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30522276

RESUMO

The next generation of acoustic sensors is emerging to supplement legacy sensors traditionally used in regional and global networks. These devices operate under similar principles as traditional sensors, without the need of a separate external digitizer. The calibration of these sensors against their predecessors is crucial to the modernization of conventional technologies. This work describes the characterization of the next-generation MB3 digital microbarometer and the iPrecision smartphone microphone in a non-isolated calibration room across the infrasound (i.e., 0.01-20 Hz) range. The intent is to evaluate nominal instrument performance before deployment. A portable rotary subwoofer is used as a controllable infrasound source to generate single-tone sinusoidal and broadband noise pressure waves in a room configured for calibration purposes. For each device, comparison measurements are made, from which the digital sensitivity and the parametric response is developed. The results provide insight into the performance of the sensors in non-isolated environments. By overlapping the responses of the test sensors, digital sensor performance across the infrasound range can be benchmarked. These responses may serve as a double-reference scheme in future pressure measurements and digital calibrations of acoustic sensors.

9.
Sci Rep ; 8(1): 13563, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30202032

RESUMO

The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Ácidos Indolacéticos/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Ensaios de Triagem em Larga Escala , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido
10.
J Agric Food Chem ; 66(24): 6123-6131, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29798665

RESUMO

We have constructed novel plasmids pANY2, pANY3, and pANY6 for flexible cloning with low false positives, efficient expression, and convenient purification of proteins. The pANY2 plasmid can be used for efficient isopropyl-ß-d-thiogalactoside (IPTG) induced protein expression, while the pANY3 plasmid can be used for temperature-induced expression. The pANY6 plasmid contains a self-cleaving elastin-like protein (ELP) tag for purification of recombinant protein by simple ELP-mediated precipitation steps and removal of the ELP tag by self-cleavage. A urea-based denaturation and refolding processes for renaturation of insoluble inclusion bodies can be conveniently integrated into the ELP-mediated precipitation protocol, removing time-consuming dialysis steps. These novel vectors, together with the described strategies of gene cloning, protein expression, and purification, may have wide applications in biosciences, agricultural, food technologies, and so forth.


Assuntos
Escherichia coli/genética , Vetores Genéticos/genética , Clonagem Molecular , Escherichia coli/metabolismo , Vetores Genéticos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Biochem Biophys Res Commun ; 497(4): 978-982, 2018 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-29448102

RESUMO

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos , Ligadura , Métodos , Plasmídeos , Reação em Cadeia da Polimerase
12.
Nat Commun ; 8(1): 1455, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29129932

RESUMO

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta-DNA complex.


Assuntos
Domínio Catalítico/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Vaccinia virus/enzimologia , Cristalografia por Raios X , DNA Glicosilases/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Nucleosídeo-Trifosfatase/genética
13.
Sci Rep ; 7(1): 3678, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623264

RESUMO

The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255-1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.


Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Correpressor 2 de Receptor Nuclear/química , Correpressor 2 de Receptor Nuclear/metabolismo , Sítios de Ligação , Expressão Gênica , Histona Desacetilases/genética , Humanos , Espectroscopia de Ressonância Magnética , Mutagênese , Correpressor 2 de Receptor Nuclear/genética , Ligação Proteica , Solubilidade , Relação Estrutura-Atividade
14.
Methods Mol Biol ; 1586: 45-63, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470598

RESUMO

Production of soluble, purifiable domains or multi-domain fragments of proteins is a prerequisite for structural biology and other applications. When target sequences are poorly annotated, or when there are few similar sequences available for alignments, identification of domains can be problematic. A method called expression of soluble proteins by random incremental truncation (ESPRIT) addresses this problem by high-throughput automated screening of tens of thousands of enzymatically truncated gene fragments. Rare soluble constructs are identified by experimental screening, and the boundaries revealed by DNA sequencing.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Biblioteca Gênica , Animais , DNA/genética , Expressão Gênica , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA/métodos , Solubilidade
15.
Sci Rep ; 7(1): 2119, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28522851

RESUMO

Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.


Assuntos
Proteínas de Ligação a Telômeros/química , Animais , Sítios de Ligação , Linhagem Celular , Camundongos , Mutação Puntual , Ligação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
16.
J Struct Biol ; 198(1): 19-27, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28268178

RESUMO

IcsA/VirG is a key virulence factor of the human pathogen Shigella flexneri, acting as both an adhesin and actin-polymerizing factor during infection. We identified a soluble expression construct of the IcsA/VirG α-domain using the ESPRIT library screening system and determined its structure to 1.9Å resolution. In addition to the previously characterized autochaperone domain, our structure reveals a new domain, which shares a common fold with the autochaperone domains of various autotransporters. We further provide insight into the previously structurally uncharacterized ß-helix domain that harbors the polar targeting motif and passenger-associated transport repeat. This structure is the first of any member of the recently identified passenger-associated transport repeat-containing autotransporters. Thus, it provides new insights into the overall architecture of this class of autotransporters, the function of the identified additional autochaperone domain and the structural properties of motifs involved in polar targeting and secretion of the Shigella flexneri virulence factor IcsA/VirG.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Shigella flexneri/patogenicidade , Fatores de Transcrição/química , Sistemas de Secreção Tipo V/metabolismo , Fatores de Virulência/química , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Estrutura Molecular , Domínios Proteicos , Transporte Proteico , Fatores de Transcrição/metabolismo
17.
J Biol Chem ; 292(16): 6468-6477, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28249940

RESUMO

Disrupted in Schizophrenia 1 (DISC1) is a scaffolding protein of significant importance for neurodevelopment and a prominent candidate protein in the pathology of major mental illness. DISC1 modulates a number of critical neuronal signaling pathways through protein-protein interactions; however, the mechanism by which this occurs and how DISC1 causes mental illness is unclear, partly because knowledge of the structure of DISC1 is lacking. A lack of homology with known proteins has hindered attempts to define its domain composition. Here, we employed the high-throughput Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) technique to identify discretely folded regions of human DISC1 via solubility assessment of tens of thousands of fragments of recombinant DISC1. We identified four novel structured regions, named D, I, S, and C, at amino acids 257-383, 539-655, 635-738, and 691-836, respectively. One region (D) is located in a DISC1 section previously predicted to be unstructured. All regions encompass coiled-coil or α-helical structures, and three are involved in DISC1 oligomerization. Crucially, three of these domains would be lost or disrupted by a chromosomal translocation event after amino acid 597, which has been strongly linked to major mental illness. Furthermore, we observed that a known illness-related frameshift mutation after amino acid 807 causes the C region to form aberrantly multimeric and aggregated complexes with an unstable secondary structure. This newly revealed domain architecture of DISC1, therefore, provides a powerful framework for understanding the critical role of this protein in a variety of devastating mental illnesses.


Assuntos
Mutação , Proteínas do Tecido Nervoso/química , Transtornos Psicóticos/genética , Esquizofrenia/genética , Mutação da Fase de Leitura , Humanos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Transdução de Sinais
18.
J Agric Food Chem ; 65(4): 867-871, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28075130

RESUMO

The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 µmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clostridium acetobutylicum/enzimologia , Hexosiltransferases/química , Hexosiltransferases/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Clostridium acetobutylicum/química , Clostridium acetobutylicum/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Frutanos/metabolismo , Hexosiltransferases/metabolismo , Cinética , Peso Molecular , Oligossacarídeos/metabolismo , Sacarose/metabolismo
19.
Front Mol Biosci ; 3: 54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27679800

RESUMO

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

20.
Protein Eng Des Sel ; 29(11): 551-556, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27578885

RESUMO

As one of the simplest and most efficient cloning methods, T-vector-based TA cloning has been widely used for cloning of single genes and construction of DNA libraries. This approach is especially suitable for high-throughput cloning of diverse DNA fragments since inserts can be cloned without knowledge of their sequence; it is therefore an ideal tool for high-throughput analysis of protein structure and function. Although most of the currently available T-vectors can only be used for cloning purposes, some novel variants with improved functions have be developed. This review focuses on recent developments of universal TA cloning methods and T-vectors constructed for function studies.

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