Specific Amino Acid Sequences Research Articles

Polymyxins Bind to the Cell Surface of Unculturable Acinetobacter baumannii and Cause Unique Dependent Resistance.

Multidrug‐resistant Acinetobacter baumannii is a top‐priority pathogen globally and polymyxins are a last‐line therapy. Polymyxin dependence in A. baumannii (i.e., nonculturable on agar without polymyxins) is a unique and highly‐resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin‐dependent A. baumannii strain possesses mutations in both lpxC (lipopolysaccharide biosynthesis) and katG (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin‐dependent growth emerges only when the lipopolysaccharide‐deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with “patch” binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol‐rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch‐binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of ‘invisible’ polymyxin‐dependent isolates in the evolution of resistance.

Illuminating the Mechanism of MHC-I Folding and Antigen Repertoire Selection Using Deep Mutational Scanning and Biophysical Studies

Abstract Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning), and selecting high affinity peptide cargo in the MHC-I groove (editing). While X-ray and cryoEM snapshots of MHC-I molecules prepared in complex with TAPBPR and Tapasin, respectively, have provided important insights into the empty MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 aa loop region in TAPBPR (corresponding to 11 residues in the sequence of Tapasin), which has been suggested to actively compete with incoming peptides by forming direct contacts with the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for its chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward editing of their peptide cargo are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner, as shown in MHC-I tetramer staining experiments using a TAPBPR library expressed on the surface of yeast. Detailed biophysical characterization by NMR and ITC reveal that the loop does not interact with the empty MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic “trap”. Our results suggest that, by utilizing a longer loop, TAPBPR lowers the affinity requirements for peptide selection relative to Tapasin to promote loading under conditions of reduced peptide concentration.

Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity.

Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The k cat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without K m change. This result indicates that combination of these mutations can further enhance TEV activity.

Artificial intelligence method to design and fold alpha-helical structural proteins from the primary amino acid sequence

The development of rational techniques to discover new mechanically relevant proteins for use in variety of applications ranging from mechanics, agriculture to biotechnology remains an outstanding nanomechanical design problem. The key barrier is to design a sequence to fold into a predictable structure to achieve a certain material function. Focused on alpha-helical proteins (as found in skin, hair, and many other mechanically relevant protein materials), we report a Multi-scale Neighborhood-based Neural Network (MNNN) model to learn how a specific amino acid sequence folds into a protein structure. The algorithm predicts the protein structure without using a template or co-evolutional information at a maximum error of 2.1 Å. We find that the prediction accuracy is higher than other models and the prediction consumes less than six orders of magnitude time than ab initio folding methods. We demonstrate that MNNN can predict the structure of an unknown protein that agrees with experiments, and our model hence shows a great advantage in the rational design of de novo proteins.

Skewed Repertoire Reconstitution and Gene Expression of HLA-A2402 CMV-CTLs after Allogeneic HCT

<h3>Background</h3> Cellular immunity is important for the control of Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplantation (HCT). However, the actual <i>in vivo</i> dynamics of CMV-specific cytotoxic T-cell (CMV-CTL) clones are still unclear. Thus, we investigated the T-cell receptor (TCR)-beta repertoire and gene expressions of HLA-A*2402-restricted CMV-CTLs in allo-HCT, using next-generation sequence methods (NGS). <h3>Patients</h3> The clone repertoire and dynamics of HLA-A*2402-restricted CMV-CTL were analyzed in 20 cases (30-1000 cells in each). First, we focused on the clone diversity in the group who had never experienced CMV reactivation after HCT from CMV-positive donors (the transferred-immunity group without CMV reactivation, n=7 cases). Next, we focused on the group who received HCT from CMV-negative donors (the naively-introduced immunity group after CMV reactivation, n=13 cases). Patients who received HCT from CMV-positive donors but experienced CMV reactivation were excluded, since there might simultaneously exist the transferred and introduced immunities. <h3>Results</h3> First, we focused on the clone diversity in the transferred immunity group (n = 7) and identified 78 clones. The top 2 clones in each case at one month after HCT exclusively accounted for >75% (range: 78-100%) of CMV-CTLs in all cases (Fig.1). In addition, these top 2 clones were also dominant in their corresponding donors (n = 3), suggesting that the transferred CMV immunity from donors would play a critical role for the prevention of CMV reactivation. Next, looking at the naively-introduced immunity group (n = 13) where 139 clones were identified, the similar skewed CMV-CTL repertoire reconstitution was also observed, and the top 2 clones accounted for >75% in 10 of the 13 cases (range: 61-100%) at 3 months after HCT (Fig.1). Focusing on the diversity of CMV-CTLs in the late phase after HCT (range: 6 -24 months, n=16), some novel clones appeared and became one of dominant clones in several cases, but the top 1 dominant clones at the early phase in each case stayed at top in 13 of the 16 cases. TRBV7 and BJ1-2 were frequently used in their CDR3 of CMV-CTLs (Fig. 2). In terms of specific amino acid sequences of CDR3 of TCR-ƒÀ, no common motif was found through all cases. However, [TSG, Q(G)GG, DPG, and NQG] motifs were frequently observed 3 months after HCT in the naively-introduced group, while [G(Q)GG or DPG] was observed even among the different cases of the transferred-immunity group. Single-cell RNA sequences demonstrated relatively uniform population in terms of gene expression even if different CMV-CTL clones existed in a patient (Fig.3). <h3>Conclusion</h3> <i>In vivo</i> clone-monitoring and gene expression of CMV-CTLs using NGS could provide an insight into the mechanism of immunological reconstitution following HCT.

Set of Approaches Based on Position Specific Scoring Matrix and Amino Acid Sequence for Primary Category Enzyme Classification

The last decade has witnessed an unprecedented accumulation of proteins in large online databases which has led to the need for automatic prediction of protein function essential for massive and timely annotations of the proteins in these datasets. Protein databases, combined with functional annotations and machine learning (ML) techniques, offer many potential benefits, including significantly facilitating rapid pharmacological target identification. The main objective of this study is to identify, for the problem of enzyme classification, the most powerful combinations of descriptors taken from different protein representations. To achieve this objective, four approaches for representing the Position-Specific Scoring Matrix (PSSM) combined with three methods for representing the Amino Acid Sequence (AAS) are evaluated with the aim of experimentally producing a powerful ensemble of descriptors for enzyme function prediction. Each protein descriptor is classified by a Support Vector Machine (SVM), with the set of SVMs finally combined by sum rule. Cross-validation experiments using these descriptors on single-functional enzymes (n=44,661) extracted from the PDB database demonstrate that the ensemble proposed here achieves superior classification rates compared to state-of-the-art ML techniques reported in the literature on the same dataset. Although the proposed ensemble strongly outperforms these other techniques, it is computationally much heavier, mainly because the PSSM extraction process is time consuming. However, there is a growing repository of proteins where PSSM has already been extracted, making the proposed method more practical and attractive. The MATLAB code and the dataset used in the experiments reported here are available at https://github.com/LorisNanni.

Biopeptides from vegetable proteins: new scientific evidences

In the last few decades, the search for new bioactive compounds able to fight several chronic degenerative diseases has increased. In this context, natural sources are especially appealing. Among these new products, peptides are included, for the promising bioactivities and potential applications. In fact, bioactive peptides represent specific sequences of amino acids with numerous health effects so they can be used for the formulation of new drugs, nutraceuticals and ingredients for functional foods. They can be obtained from different protein sources both of animal (milk, derivatives and eggs, meat, fish and even insects) and of vegetable origin (soy, wheat, corn, rice, barley, potatoes, algae). In particular, here it is shown an overview of recent data on some bioactive peptides of plant origin.

Ultrahighly Efficient and Stable Fluorescent Gold Nanoclusters Coated with Screened Peptides of Unique Sequences for Effective Protein and Serum Discrimination.

Gold nanoclusters (Au NCs) coated with various peptides have been widely used as fluorescent probes, and nowadays the most commonly used are cysteine (C) and tyrosine (Y) based ones. Herein, we report the preparation and clinical application of highly efficient and stable fluorescent Au NCs protected by screened peptides with a specific amino acid sequence Cys-Met-Met-Met-Met-Met (CMMMMM). Compared with traditional C, Y based peptide (CYYYYY) protected Au NCs, the fluorescence intensity of the CMMMMM-Au NCs increased by 230%, and the photobleaching resistance or stability of the CMMMMM-Au NCs increased by about 300% (after continuous ultraviolet irradiation for 60 min, the fluorescence of the CMMMMM-Au NCs remained more than 90% of their initial intensity, while the CYYYYY-Au NCs remained less than 30%). Assaying arrays based on CMMMMM protected Au NCs with different positive or negative charges as sensing receptors were developed through regulating different pH values, and multivariate analysis on the patterns obtained by these arrays allowed effective identification of not only ten proteins separately but also complex protein mixtures with subtly diverse compositions. The docking simulation and isothermal titration confirmed that target proteins interacted with CMMMMM-Au NCs mainly through electrostatic interactions and partly hydrophobic interactions, which affected the binding energy and fluorescence lifetime of CMMMMM-Au NCs, resulting in the unique fingerprint-like recognition patterns. Furthermore, serums from breast cancer, severe osteoarthritis, and rectal cancer patients can be effectively identified with healthy people using this CMMMMM-Au NCs based sensor array.

Cationic Antimicrobial Peptides for Tuberculosis: A Mini-Review.

Cationic antimicrobial peptides (CAMPs) can be considered as new potential therapeutic agents for Tuberculosis treatment with a specific amino acid sequence. New studies can be developed in the future to improve the pharmacological properties of CAMPs and also understand possible resistance mechanisms. This review discusses the principal properties of natural and/or synthetic CAMPs, and how these new peptides have a significant specificity for Mycobacterium tuberculosis. Also, we propose some alternative strategies to enhance the therapeutic activity of these CAMPs that include coadministration with nanoparticles and/or classic drugs.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (IK1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the “epestfind” webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical IK1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of IK1 channels.

Development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial

Collagen is the most abundant protein in the animal kingdom and has a unique triple-helical structure. It not only provides mechanical strength to tissues, but also performs specific biological functions as a multifaceted signaling molecule. Animal-derived collagen is therefore widely used as a biocompatible material in vitro and in vivo. In this study, we developed a novel peptide-based material that mimicked both the polymeric properties and a selected biological function of native collagen. This material was prepared by end-to-end multiple disulfide cross-linking of chemically synthesized triple-helical peptides. The peptide polymer showed a gel-forming property, and receptor-specific cell binding was observed in vitro by incorporating a peptide harboring an integrin α2β1-binding sequence. Furthermore, cell signaling activity and biodegradability were tunable according to the polymer contents. The results demonstrated the potential of this material as a designer collagen. Statement of SignificanceCollagen is a useful biomaterial with the gel-forming property. It also exhibits various biological activities through the interaction of specific amino acid sequences displayed on the triple helix with functional biomacromolecules. Here we report a novel synthetic material, artificial collagen, by end-to-end cross-linking of chemically synthesized collagen-like triple-helical peptides. The material allows independent regulation of polymer properties, i.e. gel stiffness, and sequence-specific bioactivities by altering peptide compositions. This material can also be variously shaped, for example, thin films with high transparency. In addition, it has low inflamatogenic properties and tunable biodegradability in vivo.

Back Cover: Identification and Quantification of Proteoforms by Mass Spectrometry

DOI: 10.1002/pmic.201800361 A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In article number 1800361, Leah V. Schaffer et al. outline recent advances and discuss current challenges and future directions for the field.

Identification and Quantification of Proteoforms by Mass Spectrometry.

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.

Mutations in the Human Follicle Stimulating Hormone Receptor Caveolin Binding Motif Cause Increased Basal Activation

Over twelve percent of women aged fifteen to forty‐five in America suffer from infertility and over seven million women have used infertility services, such as intrauterine insemination and in vitro fertilization. Some cases of infertility may be due to dysfunctional human follicle stimulating hormone (hFSH) signaling. hFSH plays a role in spermatogenesis in males, as well as follicle maturation and estrogen production in females. Problems with either hFSH or the hFSH receptor (hFSHR) decrease fertility in males and cause complete infertility in females. As part of the hypothalamic‐pituitary‐gonadal axis, hFSH is released from the pituitary gland and binds to hFSHR in the ovaries and testes. It has been shown that hFSHR interacts with lipid rafts, stiffer regions of the plasma membrane with higher sphingolipid and cholesterol concentrations, and with caveolin, a protein found in certain lipid rafts. This interaction may occur through a specific sequence of amino acids in the 4th transmembrane domain of the hFSHR consistent with a caveolin binding motif (CBM). Previous work from our lab also suggested that hFSHR signaling is regulated through residency in lipid rafts, such that hFSHR signaling is inhibited as a result of recruitment into lipid rafts. The current study investigated the effect of mutations of the CBM on hFSHR signaling. It was hypothesized that complex mutation of the CBM would cause increased hFSHR signaling by disrupting interactions with caveolin, preventing inhibition via residency in lipid rafts. Mutants were created using site‐directed mutagenesis and expression vectors were transiently transfected into HEK293 cells and hFSHR signaling was qualitatively compared between mutants via western blot. Mutation of two sites in the CBM resulted in increased basal and induced hFSHR signaling, yet mutation of three sites decreased signaling below that of the wild type receptor. Mutation of two sites supports the hypothesis, while the decreased signaling from the triple mutant may suggest too large of a conformational change to allow for any receptor function. Further understanding of hFSHR signaling regulation would allow for development of new fertility treatments for men and women.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hsp90 Recognizes a Conserved Motif in the A Chains of ADP‐Ribosylating Toxins that Move from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) enters the cell via receptor‐mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP‐ribosylation of its G‐protein target. We have shown that the cytosolic chaperone Hsp90 is required for CTA1 translocation to the cytosol. We have also shown that Hsp90 is able to independently bind and refold CTA1. Using libraries of CTA1‐derived peptides, we have identified two distinct Hsp90 binding sites within the 192 amino acid protein, an N‐terminal RPPDEI (CTA111–16) motif and a C‐terminal LDIAPA (CTA1153–158) motif. Peptides corresponding to either site were able to block Hsp90 from binding to full‐length CTA1, and both binding sites were required for translocation of CTA1 to the cytosol. The LDIAPA motif is unique to CTA1, but an RPPDEI‐like motif is present in four other ER‐translocating ADP‐ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat‐labile toxin, and Salmonella Typhimurium ADP‐ribosylating toxin. Using site‐directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90 and are essential for toxin translocation from the ER to the cytosol.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

About TFE: Old and New Findings.

The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.

Transgenic substitution with Greater Amberjack Seriola dumerili fish insulin 2 in NOD mice reduces beta cell immunogenicity

Type I diabetes (T1D) is caused by immune-mediated destruction of pancreatic beta cells. This process is triggered, in part, by specific (aa 9–23) epitopes of the insulin Β chain. Previously, fish insulins were used clinically in patients allergic to bovine or porcine insulin. Fish and human insulin differ by two amino acids in the critical immunogenic region (aa 9–23) of the B chain. We hypothesized that β cells synthesizing fish insulin would be less immunogenic in a mouse model of T1D. Transgenic NOD mice in which Greater Amberjack fish (Seriola dumerili) insulin was substituted for the insulin 2 gene were generated (mouse Ins1−/− mouse Ins2−/− fish Ins2+/+). In these mice, pancreatic islets remained free of autoimmune attack. To determine whether such reduction in immunogenicity is sufficient to protect β cells from autoimmunity upon transplantation, we transplanted fish Ins2 transgenic (expressing solely Seriola dumerili Ins2), NOD, or B16:A-dKO islets under the kidney capsules of 5 weeks old female NOD wildtype mice. The B:Y16A Β chain substitution has been previously shown to be protective of T1D in NOD mice. NOD mice receiving Seriola dumerili transgenic islet transplants showed a significant (p = 0.004) prolongation of their euglycemic period (by 6 weeks; up to 18 weeks of age) compared to un-manipulated female NOD (diabetes onset at 12 weeks of age) and those receiving B16:A-dKO islet transplants (diabetes onset at 12 weeks of age). These data support the concept that specific amino acid sequence modifications can reduce insulin immunogenicity. Additionally, our study shows that alteration of a single epitope is not sufficient to halt an ongoing autoimmune response. Which, and how many, T cell epitopes are required and suffice to perpetuate autoimmunity is currently unknown. Such studies may be useful to achieve host tolerance to β cells by inactivating key immunogenic epitopes of stem cell-derived β cells intended for transplantation.

Charge-mediated proteasome targeting.

A majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin (Ub), representing a universal degradation signal operated by the ubiquitination system. Ub-independent proteasome targeting is rationalized by the existence of 2 types of direct proteasome signals (DPSs), specific amino acid sequences or post-translational modifications, which are recognized by proteasome regulatory subunits. Historically, the first type was shown to exist in ornithine decarboxylase, whereas acetylation of core histones recently was reported as a second type of DPS. Here we declare a third type, representing charge-mediated DPS. This discovered DPS may be classified as a monopartite composition- but not sequence-dependent element of ∼70 Å in length enriched in basic and flexible amino acids. This type of degradation signal, which may be provided by cationic chemicals, is most efficiently engaged by proteasomes capped with regulator (REG)α or REGγ in an ATP-independent manner. Taken together, our findings suggest a novel modality of proteasome-substrate interrelation bypassing ubiquitination.-Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V., Belogurov, A. Charge-mediated proteasome targeting.

Lysine Malonylation Identification inE. coliwith Multiple Features

Motivation: Lysine malonylation in eukaryote proteins had been found in 2011 through high-throughput proteomic analysis. However, it was poorly understood in prokaryotes. Recent researches have shown that maonylation in E. coli was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis.Results:In this work we proposed a predictor to identify the lysine malonylation sites in E. coli through physicochemical properties, binary code and sequence frequency by support vector machine algorithm. The experimentally determined lysine malonylation sites were retrieved from the first and largest malonylome dataset in prokaryotes up to date. The physicochemical properties plus position specific amino acid sequence propensity features got the best results with AUC (the area under the Receive Operating Character curve) 0.7994, MCC (Mathew correlation coefficient) 0.4335 in 10-fold cross-validation. Meanwhile the AUC values were 0.7800, 0.7851 and 0.8050 in 6-fold, 8-fold and LOO (leave-one-out) cross-validation, respectively. All the ROC curves were close to each other which illustrated the robustness and performance of the proposed predictor. We also analyzed the sequence propensities through TwoSampleLogo and found some peptides differences with t-test p&lt;0.01. The predictor had shown better results than those of other methods K-Nearest Neighbors, C4.5 decision tree, Naïve Bayes and Random Forest. Functional analysis showed that malonylated proteins were involved in many transcription activities and diverse biological processes. Meanwhile we also developed an online package which could be freely downloaded https://github.com/Sunmile/ Malonylation E.coli.

Structural Analysis of Piezo1 Ion Channel Reveals the Relationship between Amino Acid Sequence Mutations and Human Diseases

Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piezo1 has been observed to distribute mainly in nonsensory tissues, regulating osmotic homeostasis, proprioception, and light touch. With previous studies on the putative structure of Piezo1, the gating system and several mechanotransduction mechanisms have been proposed. Besides, mutations of specific amino acid sequences in Piezo1 have been linked to several human diseases such as dehydrated hereditary xerocytosis (DHS) and congenital lymphatic dysplasia (CLD). However, most of these mutations have not been well characterized. To further elucidate the relations between these mutations and diseases, UCSF Chimera is used as the tool to visualize the structural importance of each of these mutated amino acids. With the aid from UCSF Chimera, this study has recorded and interpreted clashes and contacts originated from each of the mutations. Accordingly, specific mechanisms between mutations and human diseases are proposed, which pave the way for healing.