RESUMO
Importance: VEXAS (vacuoles, E1-ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic) syndrome is a disease with rheumatologic and hematologic features caused by somatic variants in UBA1. Pathogenic variants are associated with a broad spectrum of clinical manifestations. Knowledge of prevalence, penetrance, and clinical characteristics of this disease have been limited by ascertainment biases based on known phenotypes. Objective: To determine the prevalence of pathogenic variants in UBA1 and associated clinical manifestations in an unselected population using a genomic ascertainment approach. Design, Setting, and Participants: This retrospective observational study evaluated UBA1 variants in exome data from 163â¯096 participants within the Geisinger MyCode Community Health Initiative. Clinical phenotypes were determined from Geisinger electronic health record data from January 1, 1996, to January 1, 2022. Exposures: Exome sequencing was performed. Main Outcomes and Measures: Outcome measures included prevalence of somatic UBA1 variation; presence of rheumatologic, hematologic, pulmonary, dermatologic, and other findings in individuals with somatic UBA1 variation on review of the electronic health record; review of laboratory data; bone marrow biopsy pathology analysis; and in vitro enzymatic assays. Results: In 163â¯096 participants (mean age, 52.8 years; 94% White; 61% women), 11 individuals harbored likely somatic variants at known pathogenic UBA1 positions, with 11 of 11 (100%) having clinical manifestations consistent with VEXAS syndrome (9 male, 2 female). A total of 5 of 11 individuals (45%) did not meet criteria for rheumatologic and/or hematologic diagnoses previously associated with VEXAS syndrome; however, all individuals had anemia (hemoglobin: mean, 7.8 g/dL; median, 7.5 g/dL), which was mostly macrocytic (10/11 [91%]) with concomitant thrombocytopenia (10/11 [91%]). Among the 11 patients identified, there was a pathogenic variant in 1 male participant prior to onset of VEXAS-related signs or symptoms and 2 female participants had disease with heterozygous variants. A previously unreported UBA1 variant (c.1861A>T; p.Ser621Cys) was found in a symptomatic patient, with in vitro data supporting a catalytic defect and pathogenicity. Together, disease-causing UBA1 variants were found in 1 in 13â¯591 unrelated individuals (95% CI, 1:7775-1:23â¯758), 1 in 4269 men older than 50 years (95% CI, 1:2319-1:7859), and 1 in 26â¯238 women older than 50 years (95% CI, 1:7196-1:147â¯669). Conclusions and Relevance: This study provides an estimate of the prevalence and a description of the clinical manifestations of UBA1 variants associated with VEXAS syndrome within a single regional health system in the US. Additional studies are needed in unselected and genetically diverse populations to better define general population prevalence and phenotypic spectrum.
Assuntos
Síndromes Mielodisplásicas , Dermatopatias Genéticas , Enzimas Ativadoras de Ubiquitina , Feminino , Humanos , Masculino , Biópsia , Registros Eletrônicos de Saúde , Prevalência , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/epidemiologia , Síndromes Mielodisplásicas/genética , Enzimas Ativadoras de Ubiquitina/genética , Mutação , Estudos Retrospectivos , Exoma , Pessoa de Meia-Idade , Dermatopatias Genéticas/complicações , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/epidemiologia , Dermatopatias Genéticas/genética , Estados Unidos/epidemiologiaRESUMO
The aggregation of intrinsically disordered proteins into fibrils is implicated in many neurodegenerative diseases. Amyloid aggregation is a generic property of proteins as evidenced by globular proteins that often form amyloid aggregates under partially denaturing conditions. Recently, multiple lines of evidence have suggested that the amyloid aggregation of globular proteins can also occur under native conditions. Unfortunately, amyloid aggregation under native conditions has been demonstrated in only a handful of cases. Engineering a globular protein's amyloid aggregation might benefit from its fusion to an amyloid-derived fragment with reduced aggregation propensity. Unfortunately, the impacts of such fragments on the amyloid aggregation under native conditions have yet to be examined. In this study, we show that a globular protein, Bacillus circulans xylanase (BCX), can aggregate to form amyloid fibrils under native conditions. When BCX was mixed with or fused to the non-self-aggregating fragments, KLVFWAK and ELVFWAE-which were derived from ß-amyloid (Aß)-they modulated the BCX amyloid aggregation to differing extents. This study also provides insight into a correlation between the kinetic stability and amyloid aggregation of BCX, and supports a view that Aß-derived fragments can be useful for the modulating amyloid aggregation of some, though not all, proteins.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/química , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Estabilidade Enzimática , Cinética , Fragmentos de Peptídeos/química , Multimerização Proteica , Termodinâmica , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin-binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted ß-lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution.
Assuntos
Proteínas Arqueais/química , Pyrococcus furiosus/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Entropia , Cinética , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Circular permutation is an important protein engineering tool used to create sequence diversity of a protein by changing its linear order of amino acid sequence. Circular permutation has proven to be effective in the evolution of proteins for desired properties while maintaining similar three-dimensional structures. Due to the lack of a robust design principle guiding the selection of new termini, construction of a combinatorial library is much preferred for comprehensive evaluation of circular permutation. Unfortunately, the conventional methods used to create random circular permutation libraries cause significant sequence modification at new termini of circular permutants. In addition, these methods impose additional limitations by requiring either relatively inefficient blunt-end ligation during library construction or redesign of transposons for tailored expression of circular permutants. In this study, we present the development of an engineered transposon for facile construction of random circular permutation libraries. We provide evidence that minimal modification at the new termini of the random circular permutants is possible with our engineered transposon. In addition, our method enables the use of sticky-end ligation during library construction and provides external tunability for expression of random circular permutants.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Circular/genética , Biblioteca Gênica , Engenharia Genética/métodos , Sequência de Bases , Eletroforese em Gel de Ágar , Vetores Genéticos/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional/genética , Plasmídeos/metabolismoRESUMO
Insertional fusion between multiple protein domains represents a novel means of creating integrated functionalities. Currently, there is no robust guideline for selection of insertion sites ensuring the desired functional outcome of insertional fusion. Therefore, construction and testing of random domain insertion libraries, in which a host protein domain is randomly inserted into a guest protein domain, significantly benefit extensive exploration of sequence spaces for insertion sites. Short peptide residues are usually introduced between protein domains to alleviate structural conflicts, and the interdomain linker residues may affect the functional outcome of protein insertion complexes. Unfortunately, optimal control of interdomain linker residues is not always available in conventional methods used to construct random domain insertion libraries. Moreover, most conventional methods employ blunt-end rather than sticky-end ligation between host and guest DNA fragments, thus lowering library construction efficiency. Here, we report the facile construction of random domain insertion libraries using an engineered transposon. We show that random domain insertion with optimal control of interdomain linker residues was possible with our engineered transposon-based method. In addition, our method employs sticky-end rather than blunt-end ligation between host and guest DNA fragments, thus allowing for facile construction of relatively large sized libraries.
Assuntos
Elementos de DNA Transponíveis/genética , Biblioteca Genômica , Mutagênese Insercional/genética , Sequência de Bases , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , Transposases/metabolismoRESUMO
PURPOSE: The anatomical arrangement of the thumb pulley system continues to be revised through ongoing investigative research, changing our previous assumptions. This study demonstrates the components and anatomical features of this pulley system in an effort to improve surgical outcomes and to clarify current misconceptions. METHODS: Researchers procured 75 hand specimens from 41 adult cadavers through our institution's anatomical donations program. Dissections of the thumb and thenar compartment identified the various pulleys. A detailed analysis of the thumb pulleys was performed through various measurements. RESULTS: Four different pulley categories were identified: type I (n = 5), type II (n = 29), type III (n = 29), and type IV (n = 12). The variable annular pulley was present in 70 of 75 hands (93%) in 1 of 3 arrangements: transverse, oblique, or fused with the A1 pulley. CONCLUSIONS: The pulley system of the thumb is composed of 4 components, as opposed to the traditional view of only 3. Along with the A1 pulley, the additional variable annular pulley might contribute to stenosis in trigger thumb. This might necessitate a more extensive surgical incision and its partial release to relieve triggering. CLINICAL RELEVANCE: Understanding the anatomical configuration of the thumb pulley system will aid in the surgical attempt to resolve triggering while avoiding complications such as bowstringing of the flexor pollicis longus tendon and iatrogenic nerve injury.
Assuntos
Tendões/anatomia & histologia , Polegar/anatomia & histologia , Dedo em Gatilho/cirurgia , Cadáver , Constrição Patológica , Feminino , Humanos , Masculino , Tendões/cirurgia , Polegar/patologia , Polegar/cirurgiaRESUMO
INTRODUCTION: Pulmonary hypertension secondary to scurvy is a rare manifestation that historically has not been well studied and is only described in a handful of case reports. CASE: Our case is about a 35-year-old female with a history significant for drug and tobacco abuse, obesity, poor diet, anxiety, and major depressive disorder who was found to have severe pulmonary hypertension in the setting of vitamin C deficiency (<0.01 mg/L). CONCLUSION: We present a case that shows pulmonary hypertension can be associated with scurvy and reversed rapidly with adequate vitamin C supplementation.
RESUMO
High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.
Assuntos
Proteínas Arqueais , Bacillus/genética , Proteínas de Bactérias , Evolução Molecular Direcionada , Endo-1,4-beta-Xilanases , Temperatura Alta , Lectinas , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusão , Proteínas Arqueais/biossíntese , Proteínas Arqueais/química , Proteínas Arqueais/genética , Bacillus/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Lectinas/biossíntese , Lectinas/química , Lectinas/genética , Estabilidade Proteica , Pyrococcus furiosus/enzimologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.
Assuntos
Proteínas Arqueais/química , Bacillus/genética , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusão/química , Proteínas Arqueais/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Pyrococcus furiosus/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Protein insertional fusion and circular permutation are 2 promising protein engineering techniques for creating integrated functionalities and sequence diversity of a protein, respectively. Finding insertion locations for protein insertional fusion and new termini for circular permutation through a rational approach is not always straightforward, especially, for proteins without detailed structural knowledge. On the contrary, a combinatorial approach facilitates a comprehensive search to evaluate all potential insertion sites and new termini locations. Conventional methods used to create random insertional fusion libraries generate sub-optimal inter-domain linker length and composition between fused proteins. There are also methods available for construction of random circular permutation libraries. However, these methods too, impose many drawbacks, such as significant sequence modification at the new termini of circular permutants and additionally, require re-design of transposons for tailored expression of circular permutants. Furthermore, these conventional methods employ relatively inefficient blunt-end ligation during library construction. In this commentary, we present a concise overview and key findings of engineered Mu transposons, which have recently been developed in our group as a facile and efficient tool to alleviate limitations realized from conventional methods and to construct high quality libraries for random insertional fusion and random circular permutation.