Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the \u3e20 distinct compound classes is also reviewed, and commonalities are discussed

Characterization of the Conus bullatus genome and its venom-duct transcriptome

<p>Abstract</p> <p>Background</p> <p>The venomous marine gastropods, cone snails (genus <it>Conus</it>), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. <it>Conus bullatus </it>belongs to a clade of <it>Conus </it>species called <it>Textilia</it>, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic <it>Textilia </it>clade.</p> <p>Results</p> <p>We have carried out a sequencing survey of the <it>Conus bullatus </it>genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an <it>in silico </it>pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities.</p> <p>Conclusions</p> <p>Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of <it>Conus bullatus </it>venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of <it>C. bullatus </it>transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.</p

HLA-A and -B alleles and haplotypes in hemochromatosis probands with HFE C282Y homozygosity in central Alabama

BACKGROUND: We wanted to quantify HLA-A and -B allele and haplotype frequencies in Alabama hemochromatosis probands with HFE C282Y homozygosity and controls, and to compare results to those in other populations. METHODS: Alleles were detected using DNA-based typing (probands) and microlymphocytotoxicity (controls). RESULTS: Alleles were determined in 139 probands (1,321 controls) and haplotypes in 118 probands (605 controls). In probands, A*03 positivity was 0.7482 (0.2739 controls; p =< 0.0001; odds ratio (OR) 7.9); positivity for B*07, B*14, and B*56 was also increased. In probands, haplotypes A*03-B*07 and A*03-B*14 were more frequent (p < 0.0001, respectively; OR = 12.3 and 11.1, respectively). The haplotypes A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*35, A*03-B*44, A*03-B*47, and A*03-B*57 were also significantly more frequent in probands. 37.3% of probands were HLA-haploidentical with other proband(s). CONCLUSIONS: A*03 and A*03-B*07 frequencies are increased in Alabama probands, as in other hemochromatosis cohorts. Increased absolute frequencies of A*03-B*35 have been reported only in the present Alabama probands and in hemochromatosis patients in Italy. Increased absolute frequencies of A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*44, A*03-B*47, and A*03-B*57 in hemochromatosis cohorts have not been reported previously

Folding Circular Permutants of IL-1β: Route Selection Driven by Functional Frustration

Interleukin-1β (IL-1β) is the cytokine crucial to inflammatory and immune response. Two dominant routes are populated in the folding to native structure. These distinct routes are a result of the competition between early packing of the functional loops versus closure of the β-barrel to achieve efficient folding and have been observed both experimentally and computationally. Kinetic experiments on the WT protein established that the dominant route is characterized by early packing of geometrically frustrated functional loops. However, deletion of one of the functional loops, the β-bulge, switches the dominant route to an alternative, yet, as accessible, route, where the termini necessary for barrel closure form first. Here, we explore the effect of circular permutation of the WT sequence on the observed folding landscape with a combination of kinetic and thermodynamic experiments. Our experiments show that while the rate of formation of permutant protein is always slower than that observed for the WT sequence, the region of initial nucleation for all permutants is similar to that observed for the WT protein and occurs within a similar timescale. That is, even permutants with significant sequence rearrangement in which the functional-nucleus is placed at opposing ends of the polypeptide chain, fold by the dominant WT “functional loop-packing route”, despite the entropic cost of having to fold the N- and C- termini early. Taken together, our results indicate that the early packing of the functional loops dominates the folding landscape in active proteins, and, despite the entropic penalty of coalescing the termini early, these proteins will populate an entropically unfavorable route in order to conserve function. More generally, circular permutation can elucidate the influence of local energetic stabilization of functional regions within a protein, where topological complexity creates a mismatch between energetics and topology in active proteins

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

HFE C282Y and H63D in adults with malignancies in a community medical oncology practice

BACKGROUND: We sought to compare frequencies of HFE C282Y and H63D alleles and associated odds ratios (OR) in 100 consecutive unrelated white adults with malignancy to those in 318 controls. METHODS: Data from patients with more than one malignancy were analyzed according to each primary malignancy. For the present study, OR ≥2.0 or ≤0.5 was defined to be increased or decreased, respectively. RESULTS: There were 110 primary malignancies (52 hematologic neoplasms, 58 carcinomas) in the 100 adult patients. Allele frequencies were similar in patients and controls (C282Y: 0.0850 vs. 0.0896, respectively (OR = 0.9); H63D: 0.1400 vs. 0.1447, respectively (OR = 0.9)). Two patients had hemochromatosis and C282Y homozygosity. With C282Y, increased OR occurred in non-Hodgkin lymphoma, myeloproliferative disorders, and adenocarcinoma of prostate (2.0, 2.8, and 3.4, respectively); OR was decreased in myelodysplasia (0.4). With H63D, increased OR occurred in myeloproliferative disorders and adenocarcinomas of breast and prostate (2.4, 2.0, and 2.0, respectively); OR was decreased in non-Hodgkin lymphoma and B-chronic lymphocytic leukemia (0.5 and 0.4, respectively). CONCLUSIONS: In 100 consecutive adults with malignancy evaluated in a community medical oncology practice, frequencies of HFE C282Y or H63D were similar to those in the general population. This suggests that C282Y or H63D is not associated with an overall increase in cancer risk. However, odds ratios computed in the present study suggest that increased (or decreased) risk for developing specific types of malignancy may be associated with the inheritance of HFE C282Y or H63D. Study of more patients with these specific types of malignancies is needed to determine if trends described herein would remain and yield significant differences

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

BACKGROUND:Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. METHODOLOGY/PRINCIPAL FINDINGS:We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T-->S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. CONCLUSIONS/SIGNIFICANCE:The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features

Deciphering the Preference and Predicting the Viability of Circular Permutations in Proteins

Circular permutation (CP) refers to situations in which the termini of a protein are relocated to other positions in the structure. CP occurs naturally and has been artificially created to study protein function, stability and folding. Recently CP is increasingly applied to engineer enzyme structure and function, and to create bifunctional fusion proteins unachievable by tandem fusion. CP is a complicated and expensive technique. An intrinsic difficulty in its application lies in the fact that not every position in a protein is amenable for creating a viable permutant. To examine the preferences of CP and develop CP viability prediction methods, we carried out comprehensive analyses of the sequence, structural, and dynamical properties of known CP sites using a variety of statistics and simulation methods, such as the bootstrap aggregating, permutation test and molecular dynamics simulations. CP particularly favors Gly, Pro, Asp and Asn. Positions preferred by CP lie within coils, loops, turns, and at residues that are exposed to solvent, weakly hydrogen-bonded, environmentally unpacked, or flexible. Disfavored positions include Cys, bulky hydrophobic residues, and residues located within helices or near the protein's core. These results fostered the development of an effective viable CP site prediction system, which combined four machine learning methods, e.g., artificial neural networks, the support vector machine, a random forest, and a hierarchical feature integration procedure developed in this work. As assessed by using the hydrofolate reductase dataset as the independent evaluation dataset, this prediction system achieved an AUC of 0.9. Large-scale predictions have been performed for nine thousand representative protein structures; several new potential applications of CP were thus identified. Many unreported preferences of CP are revealed in this study. The developed system is the best CP viability prediction method currently available. This work will facilitate the application of CP in research and biotechnology

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.We thank to FCT 'Fundacao para a Ciencia e Tecnologia' (scholarship SFRH/BD/38363/2007) for providing Margarida Fernandes the grant for PhD studies

HFE MUTATIONS AND IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE

Context Alcoholic liver disease (ALD) is generally associated with iron overload, which may contribute to its pathogenesis, through increased oxidative stress and cellular damage. There are conflicting reports in literature about hemochromatosis (HFE) gene mutations and the severity of liver disease in alcoholic patients. Objectives To compare the prevalence of mutations in the hemochromatosis (HFE) gene between patients with ALD and healthy controls; to assess the relation of HFE mutations with liver iron stores and liver disease severity. Methods Liver biopsy specimens were obtained from 63 ALD patients (during routine treatment) and 52 healthy controls (during elective cholecystectomy). All individuals underwent routine liver function tests and HFE genotyping (to detect wild-type sequences and C282Y, H63D, S65C, E168Q, E168X, V59M, H63H, P160delC, Q127H, Q283P, V53M and W164X mutations). Associations between HFE mutations and risk of excessive liver iron stores, abnormal serum ferritin, liver fibrosis, or necroinflammatory activity were assessed by multivariate logistic regression analysis. Results ALD patients had significantly higher serum ferritin and transferrin saturation than controls (both P Contexto A doen&#231;a hep&#225;tica alco&#243;lica (DHA) est&#225; geralmente associada &#224; sobrecarga de ferro, que pode contribuir para a sua patog&#234;nese, atrav&#233;s do aumento do estresse oxidativo e dano celular. As descri&#231;&#245;es existentes na literatura sobre a associa&#231;&#227;o entre muta&#231;&#245;es HFE e a gravidade da DHA nem sempre s&#227;o concordantes. Objetivos Comparar a preval&#234;ncia de muta&#231;&#245;es HFE entre um grupo de pacientes com DHA e uma popula&#231;&#227;o de controle. Avaliar a rela&#231;&#227;o entre muta&#231;&#245;es HFE e os dep&#243;sitos de ferro hep&#225;tico. Avaliar se a presen&#231;a dessas muta&#231;&#245;es est&#225; associada com a gravidade da DHA. M&#233;todos Compararam-se 63 pacientes com DHA que efetuaram biopsia hep&#225;tica com 52 controles saud&#225;veis. A genotipagem HFE (wild type, C282Y, H63D, S65C, E168Q, E168X, V59M, H63H, P160delC, Q127H, Q283P, V53M, W164X) e uma avalia&#231;&#227;o laboratorial de rotina (incluindo cin&#233;tica do ferro) foram feitos em todos os indiv&#237;duos. Realizou-se regress&#227;o log&#237;stica multivariada nos casos para avaliar se a presen&#231;a de muta&#231;&#245;es HFE estava relacionada com risco aumentado de dep&#243;sitos de ferro hep&#225;tico aumentados, ferritina s&#233;rica anormal, fibrose hep&#225;tica significativa ou atividade necroinflamat&#243;ria. Resultados Os pacientes apresentaram ferritina s&#233;rica e satura&#231;&#227;o da transferrina mais elevadas que os controles, mas n&#227;o existiram diferen&#231;as significativas na distribui&#231;&#227;o de muta&#231;&#245;es HFE entre pacientes e controles. Considerando apenas os pacientes, o risco relativo de estes apresentarem pelo menos uma muta&#231;&#227;o HFE e dep&#243;sitos de ferro hep&#225;tico significativos foi de 17.23 (CI 95% 2.09-142.34, P = 0.008). Contudo, a presen&#231;a de pelo menos uma muta&#231;&#227;o HFE n&#227;o esteve associada ao risco significativamente aumentado de fibrose ou atividade necroinflamat&#243;ria significativas. O fator mais determinante para apresentar ferritina s&#233;rica acima do normal foi a presen&#231;a de alcoolismo ativo, com risco relativo de 8.87 (CI 95% 2.11-34.78, P = 0.003). Conclus&#245;es N&#227;o existiram diferen&#231;as na distribui&#231;&#227;o de muta&#231;&#245;es HFE entre pacientes com DHA e controles normais. Nos pacientes, o achado de pelo menos uma muta&#231;&#227;o HFE aumentou o risco de ter dep&#243;sitos de ferro hep&#225;tico mais elevados, mas n&#227;o para ter fibrose ou atividade necroinflamat&#243;ria significativas