ResearchPad - protein-sequencing https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Sequence-structure-function relationships in class I MHC: A local frustration perspective]]> https://www.researchpad.co/article/elastic_article_15751 Class I Major Histocompatibility Complex (MHC) binds short antigenic peptides with the help of Peptide Loading Complex (PLC), and presents them to T-cell Receptors (TCRs) of cytotoxic T-cells and Killer-cell Immunglobulin-like Receptors (KIRs) of Natural Killer (NK) cells. With more than 10000 alleles, human MHC (Human Leukocyte Antigen, HLA) is the most polymorphic protein in humans. This allelic diversity provides a wide coverage of peptide sequence space, yet does not affect the three-dimensional structure of the complex. Moreover, TCRs mostly interact with HLA in a common diagonal binding mode, and KIR-HLA interaction is allele-dependent. With the aim of establishing a framework for understanding the relationships between polymorphism (sequence), structure (conserved fold) and function (protein interactions) of the human MHC, we performed here a local frustration analysis on pMHC homology models covering 1436 HLA I alleles. An analysis of local frustration profiles indicated that (1) variations in MHC fold are unlikely due to minimally-frustrated and relatively conserved residues within the HLA peptide-binding groove, (2) high frustration patches on HLA helices are either involved in or near interaction sites of MHC with the TCR, KIR, or tapasin of the PLC, and (3) peptide ligands mainly stabilize the F-pocket of HLA binding groove.

]]>
<![CDATA[Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa]]> https://www.researchpad.co/article/Na789b0ff-1b14-409c-afa6-0c7a70fc7c42

Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.

]]>
<![CDATA[Molecular analyses and phylogeny of the herpes simplex virus 2 US9 and glycoproteins gE/gI obtained from infected subjects during the Herpevac Trial for Women]]> https://www.researchpad.co/article/5c8c193cd5eed0c484b4d241

Herpes simplex virus 2 (HSV-2) is a large double-stranded DNA virus that causes genital sores when spread by sexual contact and is a principal cause of viral encephalitis in newborns and infants. Viral glycoproteins enable virion entry into and spread between cells, making glycoproteins a prime target for vaccine development. A truncated glycoprotein D2 (gD2) vaccine candidate, recently tested in the phase 3 Herpevac Trial for Women, did not prevent HSV-2 infection in initially seronegative women. Some women who became infected experienced multiple recurrences during the trial. The HSV US7, US8, and US9 genes encode glycoprotein I (gI), glycoprotein E (gE), and the US9 type II membrane protein, respectively. These proteins participate in viral spread across cell junctions and facilitate anterograde transport of virion components in neurons, prompting us to investigate whether sequence variants in these genes could be associated with frequent recurrence. The nucleotide sequences and dN/dS ratios of the US7-US9 region from viral isolates of individuals who experienced multiple recurrences were compared with those who had had a single episode of disease. No consistent polymorphism(s) distinguished the recurrent isolates. In frequently recurring isolates, the dN/dS ratio of US7 was low while greater variation (higher dN/dS ratio) occurred in US8, suggesting conserved function of the former during reactivation. Phylogenetic reconstruction of the US7-US9 region revealed eight strongly supported clusters within the 55 U.S. HSV-2 strains sampled, which were preserved in a second global phylogeny. Thus, although we have demonstrated evolutionary diversity in the US7-US9 complex, we found no molecular evidence of sequence variation in US7-US9 that distinguishes isolates from subjects with frequently recurrent episodes of disease.

]]>
<![CDATA[Profile of the tprK gene in primary syphilis patients based on next-generation sequencing]]> https://www.researchpad.co/article/5c784fecd5eed0c484007915

Background

The highly variable tprK gene of Treponema pallidum has been acknowledged to be one of the mechanisms that causes persistent infection. Previous studies have mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing.

Methods/Principal findings

We conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each V region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1–5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed by only 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each V region was found among the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy.

Conclusions

The seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each V region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development.

]]>
<![CDATA[Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli]]> https://www.researchpad.co/article/5c5df342d5eed0c484581038

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

]]>
<![CDATA[Amino acid permeases in Cryptococcus neoformans are required for high temperature growth and virulence; and are regulated by Ras signaling]]> https://www.researchpad.co/article/5c57e680d5eed0c484ef3481

Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.

]]>
<![CDATA[Hydroxycholesterol binds and enhances the anti-viral activities of zebrafish monomeric c-reactive protein isoforms]]> https://www.researchpad.co/article/5c61b7d0d5eed0c484937fef

C-reactive proteins (CRPs) are among the faster acute-phase inflammation-responses proteins encoded by one gene (hcrp) in humans and seven genes (crp1-7) in zebrafish (Danio rerio) with importance in bacterial and viral infections. In this study, we described novel preferential bindings of 25-hydroxycholesterol (25HOCh) to CRP1-7 compared with other lipids and explored the antiviral effects of both 25HOCh and CRP1-7 against spring viremia carp virus (SVCV) infection in zebrafish. Both in silico and in vitro results confirmed the antiviral effect of 25HOCh and CRP1-7 interactions, thereby showing that the crosstalk between them differed among the zebrafish isoforms. The presence of oxidized cholesterols in human atherosclerotic plaques amplifies the importance that similar interactions may occur for vascular and/or neurodegenerative diseases during viral infections. In this context, the zebrafish model offers a genetic tool to further investigate these interactions.

]]>
<![CDATA[A multigene typing system for human adenoviruses reveals a new genotype in a collection of Swedish clinical isolates]]> https://www.researchpad.co/article/5c1d5b60d5eed0c4846eb7f3

Human adenoviruses (HAdVs) are common pathogens that can cause respiratory, gastrointestinal, urogenital, and ocular infections. They are divided into seven species containing 85 genotypes. Straightforward typing systems might help epidemiological investigations. As homologous recombination frequently shapes the evolution of HAdVs, information on a single gene is seldom sufficient to allow accurate and precise typing, and complete genome-based methods are recommended. Even so, complete genome analyses are not always easy to perform for practical reasons, and in such cases a multigene system can provide considerably more information about the strain under investigation than single-gene-based methods. Here we present a rapid, generic, multigene typing system for HAdVs based on three main deterministic regions of these viruses. Three PCR systems were used to amplify the genes encoding the DNA polymerase, the penton base hypervariable Arg-Gly-Asp-containing loop, and the hexon loop 1 (hypervariable region 1–6). Using this system, we typed 281 clinical isolates, detected members of six out of seven HAdV species (Human mastadenovirus AF), and could also detect not only divergent strains of established types but also a new recombinant strain with a previously unpublished combination of adenovirus genomes. This strain was accepted by the Human Adenovirus Working Group as a novel genotype: HAdV-86. Seven strains that could not be typed with sufficient accuracy were also investigated using a PCR based on part of the fiber gene. By analysis of corresponding sequences of the 86 known HAdV genotypes, we determined that the proposed typing system should be able to distinguish all non-recombinant types, and with additional fiber information, all known HAdV genotypes.

]]>
<![CDATA[Bridging immunogenetics and immunoproteomics: Model positional scanning library analysis for Major Histocompatibility Complex class II DQ in Tursiops truncatus]]> https://www.researchpad.co/article/5b6da1b4463d7e4dccc5faed

The Major Histocompatibility Complex (MHC) is a critical element in mounting an effective immune response in vertebrates against invading pathogens. Studies of MHC in wildlife populations have typically focused on assessing diversity within the peptide binding regions (PBR) of the MHC class II (MHC II) family, especially the DQ receptor genes. Such metrics of diversity, however, are of limited use to health risk assessment since functional analyses (where changes in the PBR are correlated to recognition/pathologies of known pathogen proteins), are difficult to conduct in wildlife species. Here we describe a means to predict the binding preferences of MHC proteins: We have developed a model positional scanning library analysis (MPSLA) by harnessing the power of mixture based combinatorial libraries to probe the peptide landscapes of distinct MHC II DQ proteins. The algorithm provided by NNAlign was employed to predict the binding affinities of sets of peptides generated for DQ proteins. These binding affinities were then used to retroactively construct a model Positional Scanning Library screen. To test the utility of the approach, a model screen was compared to physical combinatorial screens for human MHC II DP. Model library screens were generated for DQ proteins derived from sequence data from bottlenose dolphins from the Indian River Lagoon (IRL) and the Atlantic coast of Florida, and compared to screens of DQ proteins from Genbank for dolphin and three other cetaceans. To explore the peptide binding landscape for DQ proteins from the IRL, combinations of the amino acids identified as active were compiled into peptide sequence lists that were used to mine databases for representation in known proteins. The frequency of which peptide sequences predicted to bind the MHC protein are found in proteins from pathogens associated with marine mammals was found to be significant (p values <0.0001). Through this analysis, genetic variation in MHC (classes I and II) can now be associated with the binding repertoires of the expressed MHC proteins and subsequently used to identify target pathogens. This approach may be eventually applied to evaluate individual population and species risk for outbreaks of emerging diseases.

]]>
<![CDATA[Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level]]> https://www.researchpad.co/article/5b498f9c463d7e0897c6e017

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to β-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to β-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

]]>
<![CDATA[Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India]]> https://www.researchpad.co/article/5989d9f5ab0ee8fa60b6fef5

Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011–2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011–2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

]]>
<![CDATA[An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcd3f

Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

]]>
<![CDATA[Sequence Based Prediction of Antioxidant Proteins Using a Classifier Selection Strategy]]> https://www.researchpad.co/article/5989db2bab0ee8fa60bd15af

Antioxidant proteins perform significant functions in maintaining oxidation/antioxidation balance and have potential therapies for some diseases. Accurate identification of antioxidant proteins could contribute to revealing physiological processes of oxidation/antioxidation balance and developing novel antioxidation-based drugs. In this study, an ensemble method is presented to predict antioxidant proteins with hybrid features, incorporating SSI (Secondary Structure Information), PSSM (Position Specific Scoring Matrix), RSA (Relative Solvent Accessibility), and CTD (Composition, Transition, Distribution). The prediction results of the ensemble predictor are determined by an average of prediction results of multiple base classifiers. Based on a classifier selection strategy, we obtain an optimal ensemble classifier composed of RF (Random Forest), SMO (Sequential Minimal Optimization), NNA (Nearest Neighbor Algorithm), and J48 with an accuracy of 0.925. A Relief combined with IFS (Incremental Feature Selection) method is adopted to obtain optimal features from hybrid features. With the optimal features, the ensemble method achieves improved performance with a sensitivity of 0.95, a specificity of 0.93, an accuracy of 0.94, and an MCC (Matthew’s Correlation Coefficient) of 0.880, far better than the existing method. To evaluate the prediction performance objectively, the proposed method is compared with existing methods on the same independent testing dataset. Encouragingly, our method performs better than previous studies. In addition, our method achieves more balanced performance with a sensitivity of 0.878 and a specificity of 0.860. These results suggest that the proposed ensemble method can be a potential candidate for antioxidant protein prediction. For public access, we develop a user-friendly web server for antioxidant protein identification that is freely accessible at http://antioxidant.weka.cc.

]]>
<![CDATA[Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31]]> https://www.researchpad.co/article/5989daecab0ee8fa60bbf821

Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

]]>
<![CDATA[Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc68e

Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.

]]>
<![CDATA[Single-molecule protein identification by sub-nanopore sensors]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be022e

Recent advances in top-down mass spectrometry enabled identification of intact proteins, but this technology still faces challenges. For example, top-down mass spectrometry suffers from a lack of sensitivity since the ion counts for a single fragmentation event are often low. In contrast, nanopore technology is exquisitely sensitive to single intact molecules, but it has only been successfully applied to DNA sequencing, so far. Here, we explore the potential of sub-nanopores for single-molecule protein identification (SMPI) and describe an algorithm for identification of the electrical current blockade signal (nanospectrum) resulting from the translocation of a denaturated, linearly charged protein through a sub-nanopore. The analysis of identification p-values suggests that the current technology is already sufficient for matching nanospectra against small protein databases, e.g., protein identification in bacterial proteomes.

]]>
<![CDATA[Different Antibody Response against the Coxsackievirus A16 VP1 Capsid Protein: Specific or Non-Specific]]> https://www.researchpad.co/article/5989daa5ab0ee8fa60ba74f4

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease worldwide. The non-neutralizing antibody response that targets CA16 VP1 remains poorly elucidated. In the present study, antibody responses against CA16 VP1 in Shanghai blood donors and Shanxi individuals were analyzed by ELISA and inhibitory ELISA using five CA16 VP1 antigens: VP11-297, VP141-297, VP11-60, VP145-58 and VP161-297. The correlation coefficients for most of the reactions against each of the five antigens and the inhibition of the anti-CA16 VP1 antibody response produced by the various antigens were higher in Shanghai blood donors compared to those in Shanxi individuals. VP11-297 and VP141-297 strongly inhibited the anti-CA16 VP1 response in serum samples from both populations, while VP145-58 and VP161-297 intermediately and weakly inhibited the anti-CA16 VP1 response, respectively, in only Shanghai group. A specific type of inhibition (anti-CA16 VP1 was completely inhibited by both VP11-60 and VP141-297) characterized by high neutralizing antibody titers was identified and accounted for 71.4% of the strongly reactive samples from the Shanghai group. These results indicate that the Shanghai blood donors exhibited a consistent and specific antibody response, while the Shanxi individuals showed an inconsistent and non-specific antibody response. These findings may improve the understanding of host humoral immunity against CA16 and help to identify an effective approach for seroepidemiological surveillance and specific diagnosis of CA16 infection based on normal and competitive ELISA.

]]>
<![CDATA[Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo]]> https://www.researchpad.co/article/5989da07ab0ee8fa60b764a9

Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.

]]>
<![CDATA[A critical analysis of computational protein design with sparse residue interaction graphs]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd0ac

Protein design algorithms enumerate a combinatorial number of candidate structures to compute the Global Minimum Energy Conformation (GMEC). To efficiently find the GMEC, protein design algorithms must methodically reduce the conformational search space. By applying distance and energy cutoffs, the protein system to be designed can thus be represented using a sparse residue interaction graph, where the number of interacting residue pairs is less than all pairs of mutable residues, and the corresponding GMEC is called the sparse GMEC. However, ignoring some pairwise residue interactions can lead to a change in the energy, conformation, or sequence of the sparse GMEC vs. the original or the full GMEC. Despite the widespread use of sparse residue interaction graphs in protein design, the above mentioned effects of their use have not been previously analyzed. To analyze the costs and benefits of designing with sparse residue interaction graphs, we computed the GMECs for 136 different protein design problems both with and without distance and energy cutoffs, and compared their energies, conformations, and sequences. Our analysis shows that the differences between the GMECs depend critically on whether or not the design includes core, boundary, or surface residues. Moreover, neglecting long-range interactions can alter local interactions and introduce large sequence differences, both of which can result in significant structural and functional changes. Designs on proteins with experimentally measured thermostability show it is beneficial to compute both the full and the sparse GMEC accurately and efficiently. To this end, we show that a provable, ensemble-based algorithm can efficiently compute both GMECs by enumerating a small number of conformations, usually fewer than 1000. This provides a novel way to combine sparse residue interaction graphs with provable, ensemble-based algorithms to reap the benefits of sparse residue interaction graphs while avoiding their potential inaccuracies.

]]>
<![CDATA[AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis]]> https://www.researchpad.co/article/5989da7fab0ee8fa60b99e0b

Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.

]]>