Psoriasis patients are enriched for genetic variants that protect against HIV-1 disease

An important paradigm in evolutionary genetics is that of a delicate balance between genetic variants that favorably boost host control of infection but which may unfavorably increase susceptibility to autoimmune disease. Here, we investigated whether patients with psoriasis, a common immune-mediated disease of the skin, are enriched for genetic variants that limit the ability of HIV-1 virus to replicate after infection. We analyzed the HLA class I and class II alleles of 1,727 Caucasian psoriasis cases and 3,581 controls and found that psoriasis patients are significantly more likely than controls to have gene variants that are protective against HIV-1 disease. This includes several HLA class I alleles associated with HIV-1 control; amino acid residues at HLA-B positions 67, 70, and 97 that mediate HIV-1 peptide binding; and the deletion polymorphism rs67384697 associated with high surface expression of HLA-C. We also found that the compound genotype KIR3DS1 plus HLA-B Bw4-80I, which respectively encode a natural killer cell activating receptor and its putative ligand, significantly increased psoriasis susceptibility. This compound genotype has also been associated with delay of progression to AIDS. Together, our results suggest that genetic variants that contribute to anti-viral immunity may predispose to the development of psoriasis.     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.     

Individual Odors in Djungarian Hamsters (Phodopus campbelli)

The sources of scent in Djungarian hamsters (Phodopus campbelli) that may be individually discriminated were investigated using an habituation paradigm. Male Djungarian hamsters were exposed to five presentations of a particular scent from one individual, and then to the same scent from a novel individual. Increased investigation of the scent from the novel individual indicated discrimination of scents from different individuals. Male hamsters distinguished individual differences in scents of other males from the midventral gland, urine, feces, mouth, and the corner of the mouth, which includes the sacculi; they did not discriminate among odors of different individuals when the scents were from the genital region, hindfeet, fur from behind ears or fur from the back. The results indicate that Djungarian hamsters have a repertoire of individually distinctive scents that are located in specific places on the body; these scents are not actively distributed to, nor passively picked up on, other parts of the body. The fact that scents from some areas do not contain individually distinctive information suggests that some sources may be specialized for producing individually distinctive scents.     

Pyrosequencing to Identify Homogeneous Phenomenon When Using Recipients/Donors with Different CYP3A5*3 Genotypes in Living Donor Liver Transplantation

This study used pyrosequencing to determine the proportional distribution of CYP3A5*3 genotypes to further confirm the homogeneous phenomenon that is observed when recipients and donors in living donor liver transplantation (LDLT) have a different single nucleotide polymorphism (SNP) genotype. We enrolled 42 recipient/living donor pairs and the SNPs of CYP3A5*3 were identified by polymerase chain reaction-restriction fragment length polymorphism. We performed 120 liver graft biopsies as part of clinical investigations after LDLT. Pyrosequencing of the CYP3A5*3 SNPs revealed that among the 16 recipients with the G/G genotype, 94.68% had the G and 5.32% the A allele. Among the 14 recipients with the A/G genotype, 78.08% had the G and 21.92% the A allele, and among the 12 recipients with the A/A genotype, 18.45% had the G and 81.55% the A allele. Among the 12 donors with the G/G genotype, 93.85% had the G and 6.14% the A allele. Among the 26 donors with the A/G genotype, 75.73% had the G and 24.27% the A allele, and among the 4 donors with the A/A genotype, 11.09% had the G and 88.91% the A allele. There were a total of 120 liver graft biopsy samples; among the 37 recipients with the G/G genotype, 89.74% had the G and 10.26% the A allele, among the 70 recipients with the A/G genotype, 71.57% had the G and 28.43% the A allele, and among the 13 recipients with the A/A genotype, 48.25% had the G and 51.75% the A allele. The proportional distribution of G and A alleles of the CYP3A5*3 SNP between recipients/donors and liver grafts after LDLT was significantly different (p<0.001). Pyrosequencing was useful in identifying detailed proportional changes of the CYP3A5*3 SNP allele distribution, and to confirm the homogeneous phenomenon when recipients and donors in LDLT have a different genotype.     

Molecular dynamics simulations on the oligomer-formation process of the GNNQQNY peptide from yeast prion protein Sup35

Oligomeric intermediates are possible cytotoxic species in diseases associated with amyloid deposits. Understanding the early steps of fibril formation at atomic details may provide useful information for the rational therapeutic design. In this study, using the heptapeptide GNNQQNY from the yeast prion-like protein Sup35 as a model system, for which a detailed atomic structure of the fibril formed has been determined by x-ray microcrystallography, we investigated its oligomer-formation process from monomer to tetramer at the atomistic level by means of a molecular dynamics simulation with explicit water. Although the number of simulations was limited, the qualitative statistical data gave some interesting results, which indicated that the oligomer formation might start from antiparallel beta-sheet-like dimers. When a new single peptide strand was added to the preformed dimers to form trimers and then tetramers, the transition time from disorder aggregates to regular ones for the parallel alignment was found to be obviously much less than for the antiparallel one. Moreover, the parallel pattern also statistically stayed longer, providing more chances for oligomer extending, although the number of parallel stack events was almost equal to antiparallel ones. Therefore, our simulations showed that new strands might prefer to extend in a parallel arrangement to form oligomers, which agrees with the microcrystal structure of the amyloid fibril formed by this peptide. In addition, analysis of the pi-pi stacking of aromatic residues showed that this type of interaction did not play an important role in giving directionality for beta-strand alignment but played a great influence on stabilizing the structures formed in the oligomer-formation process.