Modulation of Natural HLA-B*27:05 Ligandome by Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 2 (ERAP2)

The HLA-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with AS, a chronic inflammatory spondyloarthropathy. This study examined the effect of ERAP2 in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones, and the peptides were identified using high-throughput mass spectrometry analyses. The relative abundance of a thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in the presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of the antigen-binding site of the HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and PΩ positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni. These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.

Karakteristik Fragmen Gen Penyandi Cytochrome C Oxidase Subunit I (COI) Hama Kepik Penghisap Buah Kakao (Theobroma cacao L.)

The purpose of this research is to know the character of partial sequences of the COI gene form fruit-sucking pest. The gene fragments were isolated using PCR (Polymerase Chain Reaction) techniques with specific primers, HlpF and HlpR. Character of gene fragments observed were fragment size, nucleotide sequence, similiarity, restriction map, and hydrophobicity. The size of the fragment was determined by electrophoresis of PCR products, similarity values were determined by aligning the nucleotide sequence of the PCR product with the nucleotides present in GenBank, the restriction map determined by the RestrictionMapper program, and the hydrophobicity profile determined by the BioEdit program. The results showed that PCR yield fragment size 552 pb. The results of alignment analysis showed that PCR fragment had similarity of 88% with Helopeltis theivora, 87% with Helopeltis antonii, 87% with Helopeltis bradyi and 84% with Pacipeltis maesarum. Based on the results of the analysis using RestrictionMapper program shows partial sequences of the COI gene form fruit-sucking pest has 25 sites of restriction enzyme cutting which is class of type II endonuclease enzyme. The results of the hydrophobicity analysis using the BioEdit program indicate that the COI protein is hydrophilic and hydrophobic which shows the integrated COI protein on the membrane.Keywords: COI gene fragment, fruit-sucking pest, PCR, Cocoa Crop

Characterization and Bioinformatics Analysis of C-4 Sterol Methyl Oxidase from Monascus purpureus

A gene encoding a putative C-4 sterol methyl oxidase was obtained by screening Monascus purpureus cDNA library. Bioinformatics analysis showed that this protein has a primary structure, a hydrophobicity profile and a pattern of histidine-rich motifs which are typical of C-4 methyl sterol oxidases. The deduced C-4 sterol methyl oxidase protein of M. purpureus contained 259 amino acid, with molecular mass of 30,299Da. Sequence alignment analysis revealed that M. purpureus deduced C-4 sterol methyl oxidase was closely related to C-4 sterol methyl oxidase from Aspergillus, Penicillium and Byssochlamys, and highly homologous to aforementioned and other known C-4 sterol methyl oxidase. The deduced protein is of a membrane protein with two transmembrane helices, which belongs to the fatty acid hydroxylase superfamily. The consistency of the comparison results of the primary structure, secondary structure and physicochemical properties suggests that the dedued protein may well be C-4 sterol methyl oxidase.

Modelo molecular teórico del receptor serotoninérgico 5HT2A acoplado a proteína G

<strong>Objective</strong> Build a theoretical molecular model of the tertiary structure of the Homo sapiens 5HT2A receptor from experimentally obtained structures as templates. <strong>Materials</strong> <strong>and methods</strong> In the construction of the theoretical model we considered the protocol established by Ballesteros and Weinstein for the construction of the G-protein coupled receptor, by the alignment of the amino acid sequence, hydrophobicity profiles, refinement of loops by spatial restrictions and energy minimization with the force field OPLS_2005. <strong>Results</strong> The resulting model was validated by the Ramachandran plot with 91.7% of amino acids within the limits set for angles phi and psi and a RMSD of 0.95 Å with respect to bovine rhodopsin. <strong>Conclusions</strong> We obtained a validated theoretical model useful in studies of ligand-receptor docking.<br /><strong>Key words</strong>: G protein receptor, hydrophobicity profile, Ramachandran plot, orthosteric site, molecular modelling.

Comparison of predicted binders in Rhipicephalus (Boophilus) microplus intestine protein variants BM86 Campo Grande strain, BM86 and BM95

This paper reports the sequence analysis of Bm86 Campo Grande strain comparing it with Bm86 and Bm95 antigens from the preparations TickGardPLUS and GavacTM, respectively. The PCR product was cloned into pMOSBlue and sequenced. The secondary structure prediction tool PSIPRED was used to calculate alpha helices and beta strand contents of the predicted polypeptide. The hydrophobicity profile was calculated using the algorithms from the Hopp and Woods method, in addition to identification of potential MHC class-I binding regions in the antigens. Pair-wise alignment revealed that the similarity between Bm86 Campo Grande strain and Bm86 is 0.2% higher than that between Bm86 Campo Grande strain and Bm95 antigens. The identities were 96.5% and 96.3% respectively. Major suggestive differences in hydrophobicity were predicted among the sequences in two specific regions.

Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain

ABSTRACT The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.

Site-saturation mutagenesis of the PALTAVETG motif in coxsackievirus A9 capsid protein VP1 reveals evidence of conservation of a periodic hydrophobicity profile

Enteroviruses possess a highly conserved 9 amino acid stretch of mainly hydrophobic character in the capsid protein VP1. A novel strategy, combining site-saturation mutagenesis and a single-tube cloning and transfection procedure, has been developed for the analysis of this motif in coxsackievirus A9 (CAV-9). Four individual amino acids were separately mutated. Mutagenesis of three of the four positions in CAV-9 resulted in a number of viable but impaired mutant strains, each containing a single amino acid substitution. In contrast, no mutants with amino acid substitutions at leucine 31 were isolated, although three different leucine codons were found among the viruses recovered. Small plaque size was regularly associated with reduced yields of infectious virus and an amino acid substitution at the target site in the viruses isolated from the site-saturated virus pools. From the range of amino acids observed in viable mutants, it was possible to estimate the characteristics that are required at individual amino acid positions. It seems that in the motif studied here, a periodic hydrophobicity profile needs to be conserved. The constraints observed on the ranges of acceptable amino acids presumably reflect the structural–functional requirements that have resulted in the conservation of the motif.