Two minimal Tat translocases in Bacillus Jongbloed, Jan D. H.; Grieger, Ulrike; Antelmann, Haike ...
Molecular microbiology,
December 2004, Letnik:
54, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Summary
Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: ...TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram‐positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB‐like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate‐specific translocases for the twin‐arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram‐positive bacteria, Streptomycetes being the only known exception with TatABC translocases.
Recent advancements in next generation sequencing (NGS) technology have led to the identification of the giant sarcomere gene, titin (TTN), as a major human disease gene. Truncating variants of TTN ...(TTNtv) especially in the A-band region account for 20% of dilated cardiomyopathy (DCM) cases. Much attention has been focused on assessment and interpretation of TTNtv in human disease; however, missense and non-frameshifting insertions/deletions (NFS-INDELs) are difficult to assess and interpret in clinical diagnostic workflow. Targeted sequencing covering all exons of TTN was performed on a cohort of 530 primary DCM patients from three cardiogenetic centres across Europe. Using stringent bioinformatic filtering, twenty-nine and two rare TTN missense and NFS-INDELs variants predicted deleterious were identified in 6.98% and 0.38% of DCM patients, respectively. However, when compared with those identified in the largest available reference population database, no significant enrichment of such variants was identified in DCM patients. Moreover, DCM patients and reference individuals had comparable frequencies of splice-region missense variants with predicted splicing alteration. DCM patients and reference populations had comparable frequencies of rare predicted deleterious TTN missense variants including splice-region missense variants suggesting that these variants are not independently causative for DCM. Hence, these variants should be classified as likely benign in the clinical diagnostic workflow, although a modifier effect cannot be excluded at this stage.
ABSTRACT
Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by myocardial atrophy, fibro‐fatty replacement, and a high risk of ventricular arrhythmias that lead to ...sudden death. In 2009, genetic data from 57 publications were collected in the arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) Genetic Variants Database (freeware available at http://www.arvcdatabase.info), which comprised 481 variants in eight ACM‐associated genes. In recent years, deep genetic sequencing has increased our knowledge of the genetics of ACM, revealing a large spectrum of nucleotide variations for which pathogenicity needs to be assessed. As of April 20, 2014, we have updated the ARVD/C database into the ARVD/C database to contain more than 1,400 variants in 12 ACM‐related genes (PKP2, DSP, DSC2, DSG2, JUP, TGFB3, TMEM43, LMNA, DES, TTN, PLN, CTNNA3) as reported in more than 160 references. Of these, only 411 nucleotide variants have been reported as pathogenic, whereas the significance of the other approximately 1,000 variants is still unknown. This comprehensive collection of ACM genetic data represents a valuable source of information on the spectrum of ACM‐associated genes and aims to facilitate the interpretation of genetic data and genetic counseling.
The updated www.arvcdatabase.info is a valuable and freely accessible source of information for doctors, researchers, and patients, containing clinical and genetic data on all genes associated with arrhythmogenic cardiomyopathy. The 2014 update contains more than 1,400 variants in 12 genes (PKP2, DSP, DSC2, DSG2, JUP, TGFB3, TMEM43, LMNA, DES, TTN, PLN, CTNNA3), reported in more than 160 references. Of these, 411 nucleotide variants have been reported as pathogenic.
ABSTRACT
Introduction: Centronuclear myopathies (CNMs) are a subtype of congenital myopathies (CMs) characterized by muscle weakness, predominant type 1 fibers, and increased central nuclei. SPEG ...(striated preferentially expressed protein kinase) mutations have recently been identified in 7 CM patients (6 with CNMs). We report 2 additional patients with SPEG mutations expanding the phenotype and evaluate genotype–phenotype correlations associated with SPEG mutations.
Methods: Using whole exome/genome sequencing in CM families, we identified novel recessive SPEG mutations in 2 patients.
Results: Patient 1, with severe muscle weakness requiring respiratory support, dilated cardiomyopathy, ophthalmoplegia, and findings of nonspecific CM on muscle biopsy carried a homozygous SPEG mutation (p.Val3062del). Patient 2, with milder muscle weakness, ophthalmoplegia, and CNM carried compound heterozygous mutations (p.Leu728Argfs*82) and (p.Val2997Glyfs*52).
Conclusions: The 2 patients add insight into genotype–phenotype correlations of SPEG‐associated CMs. Clinicians should consider evaluating a CM patient for SPEG mutations even in the absence of CNM features. Muscle Nerve 59:357–362, 2019
ABSTRACT
Next‐generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS‐based tests according to diagnostic standards is a ...challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of “diagnostic yield” as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete “diagnostic routing.” We also propose that laboratories should use a list of “core disease genes” for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.
One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This ...has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.
RATIONALE:Orthostatic hypotension is a common clinical problem, but the underlying mechanisms have not been fully delineated.
OBJECTIVE:We describe 2 families, with 4 patients in total, experiencing ...severe life-threatening orthostatic hypotension because of a novel cause.
METHODS AND RESULTS:As in dopamine β-hydroxylase deficiency, concentrations of norepinephrine and epinephrine in the patients were low. Plasma dopamine β-hydroxylase activity, however, was normal, and the DBH gene had no mutations. Molecular genetic analysis was performed to determine the underlying genetic cause. Homozygosity mapping and exome and Sanger sequencing revealed pathogenic homozygous mutations in the gene encoding cytochrome b561 (CYB561); a missense variant c.262G>A, p.Gly88Arg in exon 3 in the Dutch family and a nonsense mutation (c.131G>A, p.Trp44*) in exon 2 in the American family. Expression of CYB561 was investigated using RNA from different human adult and fetal tissues, transcription of RNA into cDNA, and real-time quantitative polymerase chain reaction. The CYB561 gene was found to be expressed in many human tissues, in particular the brain. The CYB561 protein defect leads to a shortage of ascorbate inside the catecholamine secretory vesicles leading to a functional dopamine β-hydroxylase deficiency. The concentration of the catecholamines and downstream metabolites was measured in brain and adrenal tissue of 6 CYB561 knockout mice (reporter-tagged deletion allele post-Cre, genetic background C57BL/6NTac). The concentration of norepinephrine and normetanephrine was decreased in whole-brain homogenates of the CYB561 mice compared with wild-type mice (P<0.01), and the concentration of normetanephrine and metanephrine was decreased in adrenal glands (P<0.01), recapitulating the clinical phenotype. The patients responded favorably to treatment with L-dihydroxyphenylserine, which can be converted directly to norepinephrine.
CONCLUSIONS:This study is the first to implicate cytochrome b561 in disease by showing that pathogenic mutations in CYB561 cause an as yet unknown disease in neurotransmitter metabolism causing orthostatic hypotension.
Aims
Truncating titin mutations (tTTN) occur in 25% of dilated cardiomyopathy (DCM) cases, but the phenotype and severity of disease they cause have not yet been systematically studied. We studied ...whether tTTN variants are associated with a clinically distinguishable form of DCM.
Methods and results
We compared clinical data on DCM probands and relatives with a tTTN mutation (n = 45, n = 73), LMNA mutation (n = 28, n = 29), and probands who tested negative for both genes idiopathic DCM (iDCM); n = 60. Median follow‐up was at least 2.5 years in each group. TTN subjects presented with DCM at higher age than LMNA subjects (probands 47.9 vs. 40.4 years, P = 0.004; relatives 59.8 vs. 47.0 years, P = 0.01), less often developed LVEF <35% probands hazard ratio (HR) 0.38, P = 0.002, had higher age of death (probands 70.4 vs. 59.4 years, P < 0.001; relatives 74.1 vs. 58.4 years, P = 0.008), and had better composite outcome (malignant ventricular arrhythmia, heart transplantation, or death; probands HR 0.09, P < 0.001; relatives HR 0.21, P = 0.02) than LMNA subjects and iDCM subjects (HR 0.36, P = 0.07). An LVEF increase of at least 10% occurred in 46.9% of TTN subjects after initiation of standard heart failure treatment, while this only occurred in 6.5% of LMNA subjects (P < 0.001) and 18.5% of iDCM subjects (P = 0.02). This was confirmed in families with co‐segregation, in which the 10% point LVEF increase occurred in 55.6% of subjects (P = 0.003 vs. LMNA, P = 0.079 vs. iDCM).
Conclusions
This study shows that tTTN‐associated DCM is less severe at presentation and more amenable to standard therapy than LMNA mutation‐induced DCM or iDCM.
RATIONALE:Idiopathic dilated cardiomyopathy (DCM) is inherited in approximately one third of cases, usually as an autosomal dominant trait. More than 30 loci have been identified, several of which ...encode sarcomeric proteins which can also be mutated to cause hypertrophic cardiomyopathy. One contractile protein gene well known as a hypertrophic cardiomyopathy disease gene, but with no reported mutation in autosomal dominant DCM, is TNNI3 which encodes cardiac troponin I.
OBJECTIVE:To test TNNI3 as a candidate gene, a panel of 96 probands with DCM was analyzed.
METHODS AND RESULTS:Genomic DNA was isolated and TNNI3 exons screened by heteroduplex analysis. Exons with aberrant profiles were sequenced and variants evaluated by segregation analysis and study of normal controls. We report 2 novel TNNI3 missense mutations, Lys36Gln and Asn185Lys, each associated with severe and early onset familial DCM. Of the 5 mutation carriers, cardiac transplantation was required in 3, at ages 6, 15, and 24 years. Analysis of Ca regulation of actin-tropomyosin–activated myosin ATPase by troponin revealed that troponin reconstituted with either mutant troponin I gave lower maximum ATPase rates and lower Ca sensitivity than wild type. Furthermore, mutant thin filaments had reduced Ca affinity compared with normal.
CONCLUSIONS:The functional alterations mirror closely a consistent phenotype found in proven DCM mutations in other thin filament proteins, thus supporting the interpretation that these mutations are disease-causing. These are the first reported autosomal dominant DCM-causing mutations in TNNI3, and so the findings expand the spectrum of disease-causing genes that lead to either hypertrophic cardiomyopathy or DCM depending on the specific mutation.
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