Amino Acid Preferences Research Articles

Functional expression and characterization of a novel aminopeptidase B from Aspergillus niger in Pichia pastoris.

A novel aminopeptidase B (APB-AN) was identified from Aspergillus niger CGMCC 3.1454 for the first time and was cloned and expressed in Pichia pastoris. The mature enzyme of approximately 100kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40°C, respectively. The enzyme was stable below 40°C and at pH values from 5.0 to 8.0. The K m and V max values were determined to be 0.61mmol/L and 11.45mmol/L/min, respectively, using Arg-pNA as the substrate. APB-AN was inhibited by Cu2+ and Fe2+ and activated by Co2+ and Na+. Most metal chelators (Ca2+, Mg2+ and Mn2+) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid p-nitroanilide (pNA) substrates:Arg-pNA, Lys-pNA, Tyr- pNA, Trp-pNA, Phe-pNA, His-pNA, Ala-pNA, Met-pNA, Leu-pNA, Glu-pNA, Val-pNA, Pro-pNA and Ile-pNA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications.

Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding

Meprinβ is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprinβ including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprinβ is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprinβ including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprinβ with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprinβ from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprinβ was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprinβ phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprinβ activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprinβ.

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein.

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

Differential Impacts of Water Table and Temperature on Bacterial Communities in Pore Water From a Subalpine Peatland, Central China.

The level of water table and temperature are two environmental variables shaping soil bacterial communities, particularly in peatland ecosystems. However, discerning the specific impact of these two factors on bacterial communities in natural ecosystems is challenging. To address this issue, we collected pore water samples across different months (August and November in 2017 and May 2018) with a gradient of water table changes and temperatures at the Dajiuhu peatland, Central China. The samples were analyzed with 16S rRNA high-throughput sequencing and Biolog EcoMicroplates. Bacterial communities varied in the relative abundances of dominant taxa and harbored exclusive indicator operational taxonomic units across the different months. Despite these differences, bacterial communities showed high similarities in carbon utilization, with preferences for esters (pyruvic acid methyl ester, Tween 40, Tween 80, and D-galactonic acid γ-lactone), amino acids (L-arginine and L-threonine), and amines (phenylethylamine and putrescine). However, rates of carbon utilization (as indicated by average well-color development) and metabolic diversity (McIntosh and Shannon index) in May and August were higher than those in November. Redundancy analysis revealed that the seasonal variations in bacterial communities were significantly impacted by the level of the water table, whereas the temperature had a fundamental role in bacterial carbon utilization rate. Co-occurrence analysis identified Sphingomonas, Mucilaginibacter, Novosphingobium, Lacunisphaera, Herminiimonas, and Bradyrhizobium as keystone species, which may involve in the utilization of organic compounds such as amino acids, phenols, and others. Our findings suggest that bacterial community functions were more stable than their compositions in the context of water table changes. These findings significantly expand our current understanding of the variations of bacterial community structures and metabolic functions in peatland ecosystems in the context of global warming and fluctuation of the water table.

Investigation on the characteristics and mechanisms of ACE inhibitory peptides by a thorough analysis of all 8000 tripeptides via binding free energy calculation.

Food‐derived angiotensin I‐converting enzyme (ACE) inhibitory peptides represent a potential source of new antihypertensive. However, their characteristics and binding mechanisms were not well understood. In this study, novel energy calculation and experimentation were combined to elucidate the characteristics and mechanisms of ACE inhibitory tripeptides. ACE inhibitory activity of all 8,000 tripeptides was investigated by in silico experiments. IC50 values of the five top‐rated tripeptides ranged from 5.86 to 21.84 μM. Five hundred top‐ranked tripeptides were chosen for detailed structure–activity analysis, and a significant preference for aromatic amino acids at both C‐ and N‐terminus was found. By binding free energy analysis of nine representative tripeptides via MM/GBSA, electrostatic energy was found to be the leading energy that contributed to the binding of ACE with its high affinity tripeptides. Besides, S355, V380, and V518, three residues positioned around the classical binding pockets of ACE, also played a key role in ACE's binding. Therefore, for tripeptides, their binding pockets in ACE were redefined. In conclusion, the characteristics of ACE inhibitory peptides were more deeply illustrated by the thorough analysis of all tripeptides. The energy analysis allows a better understanding of the binding mechanisms of ACE inhibitory peptides, which could be used to redesign the ACE inhibitors for stronger inhibitory activity.

Intrinsic Conformational Preferences of the Charged Amino Acids in Disordered Proteins

Conformational equilibria in the unfolded states of proteins have many important biological roles, from regulating how some proteins fold and maintain structural stability, to describing the macromolecular details of intrinsically disordered proteins (IDPs), a protein class that does not adopt physiologically stable structures and instead utilizes unfolded states to facilitate their functions. Recently, to better understand the structural properties of IDPs, we analyzed both the sequence dependence to the mean hydrodynamic size of IDPs in water, as well as the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the polyproline II (PPII) backbone conformation could be obtained. This result is important because PPII is used biologically for protein–protein and protein–nucleic acid interactions and thus has a role in molecular recognition. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the IDP revealed amino acid-specific preferences for PPII that were in excellent quantitative agreement with calorimetry-measured values from small unfolded peptides and also those inferred by a survey of the protein coil library. Here, a set of charge-substitution experiments were used to measure the intrinsic preferences of the charged amino acids (i.e., lysine, arginine, glutamic acid, and aspartic acid) for the PPII backbone conformation, the results of which provide insight into how long-range charge-charge interactions and short-range effects, such as hydration and sterics, can modulate the structural and dynamical properties of IDPs, an increasingly important protein class in cell function.

Thiazole\u2013amino acids: influence of thiazole ring on conformational properties of amino acid residues

Post-translational modified thiazole–amino acid (Xaa–Tzl) residues have been found in macrocyclic peptides (e.g., thiopeptides and cyanobactins), which mostly inhibit protein synthesis in Gram + bacteria. Conformational study of the series of model compounds containing this structural motif with alanine, dehydroalanine, dehydrobutyrine and dehydrophenylalanine were performed using DFT method in various environments. The solid-state crystal structure conformations of thiazole–amino acid residues retrieved from the Cambridge Structural Database were also analysed. The studied structural units tend to adopt the unique semi-extended β2 conformation; which is stabilised mainly by N–H⋯NTzl hydrogen bond, and for dehydroamino acids also by π-electron conjugation. The conformational preferences of amino acids with a thiazole ring were compared with oxazole analogues and the role of the sulfur atom in stabilising the conformations of studied peptides was discussed.

Dynamic Responses of Barley Root Succinyl-Proteome to Short-Term Phosphate Starvation and Recovery.

Barley (Hordeum vulgare L.)—a major cereal crop—has low Pi demand, which is a distinct advantage for studying the tolerance mechanisms of phosphorus deficiency. We surveyed dynamic protein succinylation events in barley roots in response to and recovery from Pi starvation by firstly evaluating the impact of Pi starvation in a Pi-tolerant (GN121) and Pi-sensitive (GN42) barley genotype exposed to long-term low Pi (40 d) followed by a high-Pi recovery for 10 d. An integrated proteomics approach involving label-free, immune-affinity enrichment, and high-resolution LC-MS/MS spectrometric analysis was then used to quantify succinylome and proteome in GN121 roots under short-term Pi starvation (6, 48 h) and Pi recovery (6, 48 h). We identified 2,840 succinylation sites (Ksuc) across 884 proteins; of which, 11 representative Ksuc motifs had the preferred amino acid residue (lysine). Furthermore, there were 81 differentially abundant succinylated proteins (DFASPs) from 119 succinylated sites, 83 DFASPs from 110 succinylated sites, 93 DFASPs from 139 succinylated sites, and 91 DFASPs from 123 succinylated sites during Pi starvation for 6 and 48 h and during Pi recovery for 6 and 48 h, respectively. Pi starvation enriched ribosome pathways, glycolysis, and RNA degradation. Pi recovery enriched the TCA cycle, glycolysis, and oxidative phosphorylation. Importantly, many of the DFASPs identified during Pi starvation were significantly overexpressed during Pi recovery. These results suggest that barley roots can regulate specific Ksuc site changes in response to Pi stress as well as specific metabolic processes. Resolving the metabolic pathways of succinylated protein regulation characteristics will improve phosphate acquisition and utilization efficiency in crops.

Candidatus Eremiobacterota, a metabolically and phylogenetically diverse terrestrial phylum with acid-tolerant adaptations.

Candidatus phylum Eremiobacterota (formerly WPS-2) is an as-yet-uncultured bacterial clade that takes its name from Ca. Eremiobacter, an Antarctic soil aerobe proposed to be capable of a novel form of chemolithoautotrophy termed atmospheric chemosynthesis, that uses the energy derived from atmospheric H2-oxidation to fix CO2 through the Calvin-Benson-Bassham (CBB) cycle via type 1E RuBisCO. To elucidate the phylogenetic affiliation and metabolic capacities of Ca. Eremiobacterota, we analysed 63 public metagenome-assembled genomes (MAGs) and nine new MAGs generated from Antarctic soil metagenomes. These MAGs represent both recognized classes within Ca. Eremiobacterota, namely Ca. Eremiobacteria and UBP9. Ca. Eremiobacteria are inferred to be facultatively acidophilic with a preference for peptides and amino acids as nutrient sources. Epifluorescence microscopy revealed Ca. Eremiobacteria cells from Antarctica desert soil to be coccoid in shape. Two orders are recognized within class Ca. Eremiobacteria: Ca. Eremiobacterales and Ca. Baltobacterales. The latter are metabolically versatile, with individual members having genes required for trace gas driven autotrophy, anoxygenic photosynthesis, CO oxidation, and anaerobic respiration. UBP9, here renamed Ca. Xenobia class. nov., areinferred to be obligate heterotrophs with acidophilic adaptations, but individual members having highly divergent metabolic capacities compared to Ca. Eremiobacteria, especially with regard to respiration and central carbon metabolism. We conclude Ca. Eremiobacterota to be an ecologically versatile phylum with the potential to thrive under an array of "extreme" environmental conditions.

Comparative Mitogenomics in Hyalella (Amphipoda: Crustacea).

We present the sequencing and comparative analysis of 17 mitochondrial genomes of Nearctic and Neotropical amphipods of the genus Hyalella, most from the Andean Altiplano. The mitogenomes obtained comprised the usual 37 gene-set of the metazoan mitochondrial genome showing a gene rearrangement (a reverse transposition and a reversal) between the North and South American Hyalella mitogenomes. Hyalella mitochondrial genomes show the typical AT-richness and strong nucleotide bias among codon sites and strands of pancrustaceans. Protein-coding sequences are biased towards AT-rich codons, with a preference for leucine and serine amino acids. Numerous base changes (539) were found in tRNA stems, with 103 classified as fully compensatory, 253 hemi-compensatory and the remaining base mismatches and indels. Most compensatory Watson–Crick switches were AU -> GC linked in the same haplotype, whereas most hemi-compensatory changes resulted in wobble GU and a few AC pairs. These results suggest a pairing fitness increase in tRNAs after crossing low fitness valleys. Branch-site level models detected positive selection for several amino acid positions in up to eight mitochondrial genes, with atp6 and nad5 as the genes displaying more sites under selection.

Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases.

The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3 (HTLV-1, -2, and -3) proteases. Amino acid preferences of S4 to S1′ subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors (atazanavir, darunavir, DMP-323, indinavir, ritonavir, and saquinavir) on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors.

Analysis of proteolytic processing sites in potyvirus polyproteins revealed differential amino acid preferences of NIa-Pro protease in each of seven cleavage sites.

Potyviruses encode a large polyprotein that undergoes proteolytic processing, producing 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb-RdRp, and CP. While P1/HC-Pro and HC-Pro/P3 junctions are cleaved by P1 and HC-Pro, respectively, the remaining seven are processed by NIa-Pro. In this study, we analyzed 135 polyprotein sequences from approved potyvirus species and deduced the consensus amino acid residues at five positions (from −4 to +1, where a protease cleaves between −1 and +1) in each of nine cleavage sites. In general, the newly deduced consensus sequences were consistent with the previous ones. However, seven NIa-Pro cleavage sites showed distinct amino acid preferences despite being processed by the same protease. At position −2, histidine was the dominant amino acid residue in most cleavage sites (57.8–60.7% of analyzed sequences), except for the NIa-Pro/NIb-RdRp junction where it was absent. At position −1, glutamine was highly dominant in most sites (88.2–97.8%), except for the VPg/NIa-Pro junction where glutamic acid was found in all the analyzed proteins (100%). At position +1, serine was the most abundant residue (47.4–86.7%) in five out of seven sites, while alanine (52.6%) and glycine (82.2%) were the most abundant in the P3/6K1 and 6K2/VPg junctions, respectively. These findings suggest that each NIa-Pro cleavage site is finely tuned for differential characteristics of proteolytic reactions. The newly deduced consensus sequences may be useful resources for the development of models and methods to accurately predict potyvirus polyprotein processing sites.

A systematic analysis of the beta hairpin motif in the Protein Data Bank.

The beta hairpin motif is a ubiquitous protein structural motif that can be found in molecules across the tree of life. This motif, which is also popular in synthetically designed proteins and peptides, is known for its stability and adaptability to broad functions. Here, we systematically probe all 49,000 unique beta hairpin substructures contained within the Protein Data Bank (PDB) to uncover key characteristics correlated with stable beta hairpin structure, including amino acid biases and enriched interstrand contacts. We find that position specific amino acid preferences, while seen throughout the beta hairpin structure, are most evident within the turn region, where they depend on subtle turn dynamics associated with turn length and secondary structure. We also establish a set of broad design principles, such as the inclusion of aspartic acid residues at a specific position and the careful consideration of desired secondary structure when selecting residues for the turn region, that can be applied to the generation of libraries encoding proteins or peptides containing beta hairpin structures.

Comparative genomics and molecular adaptational analysis of Arthrobacter from Sikkim Himalaya provided insights into its survivability under multiple high-altitude stress

Arthrobacter is a dominant aerobic bacterium under the class Actinobacteria, known for its nutritionally versatile nature and wide prevalence in stressful environments. In the current study representative two strains of Arthrobacter, ERGS1:01 and ERGS4:06, with efficient survivability under high altitude stress conditions were selected for comparative genomic studies with their mesophilic counterparts. Physiological analysis and genome insights supported the survival of these strains under multiple high-altitude stress conditions. Molecular cold-adaptation and substitution analysis of the studied strains supported the incidence of more cold-adapted proteins for functionality at low temperatures. Studied strains preferred amino acids like serine, asparagine, lysine, tryptophan for favoring increased flexibility supporting their broad temperature survivability. To the best of our knowledge, this is the first molecular cold adaptation analysis performed for the genus Arthrobacter and has revealed that ‘aromaticity’, one of the cold-adaptor indicators, should be carefully considered while evaluating cold adaptation strategies in psychrotrophic/psychrophilic bacteria.

Brassicaceae transcriptomes reveal convergent evolution of super-accumulation of sinigrin

Wasabi, horseradish and mustard are popular pungent crops in which the characteristic bioactive hydrolysis of specialized glucosinolates (GSLs) occurs. Although the metabolic pathways of GSLs are well elucidated, how plants have evolved convergent mechanisms to accumulate identical GSL components remains largely unknown. In this study, we discovered that sinigrin is predominantly synthesized in wasabi, horseradish and mustard in Brassicaceae. We de novo assembled the transcriptomes of the three species, revealing the expression patterns of gene clusters associated with chain elongation, side chain modification and transport. Our analysis further revealed that several gene clusters were convergently selected during evolution, exhibiting convergent shifts in amino acid preferences in mustard, wasabi and horseradish. Collectively, our findings provide insights into how unrelated crop species evolve the capacity for sinigrin super-accumulation and thus promise a potent strategy for engineering metabolic pathways at multiple checkpoints to fortify bioactive compounds for condiment or pharmaceutical purposes.

Peptide Binding to HLA-E Molecules in Humans, Nonhuman Primates, and Mice Reveals Unique Binding Peptides but Remarkably Conserved Anchor Residues.

Ag presentation via the nonclassical MHC class Ib molecule HLA-E, with nearly complete identity between the two alleles expressed in humans, HLA-E*01:01 and HLA-E*01:03, can lead to the activation of unconventional T cells in humans. Despite this virtual genetic monomorphism, differences in peptide repertoires binding to the two allelic variants have been reported. To further dissect and compare peptide binding to HLA-E*01:01 and HLA-E*01:03, we used an UV-mediated peptide exchange binding assay and an HPLC-based competition binding assay. In addition, we investigated binding of these same peptides to Mamu-E, the nonhuman primate homologue of human HLA-E, and to the HLA-E-like molecule Qa-1b in mice. We next exploited the differences and homologies in the peptide binding pockets of these four molecules to identify allele specific as well as common features of peptide binding motifs across species. Our results reveal differences in peptide binding preferences and intensities for each human HLA-E variant compared with Mamu-E and Qa-1b Using extended peptide libraries, we identified and refined the peptide binding motifs for each of the four molecules and found that they share main anchor positions, evidenced by conserved amino acid preferences across the four HLA-E molecules studied. In addition, we also identified differences in peptide binding motifs, which could explain the observed variations in peptide binding preferences and affinities for each of the four HLA-E-like molecules. Our results could help with guiding the selection of candidate pathogen-derived peptides with the capacity to target HLA-E-restricted T cells that could be mobilized in vaccination and immunotherapeutic strategies.

Extended Cleavage Specificity of the Rat Vascular Chymase, a Potential Blood Pressure Regulating Enzyme Expressed by Rat Vascular Smooth Muscle Cells

Serine proteases constitute the major protein content of the cytoplasmic granules of several hematopoietic cell lineages. These proteases are encoded from four different loci in mammals. One of these loci, the chymase locus, has in rats experienced a massive expansion in the number of functional genes. The human chymase locus encodes 4 proteases, whereas the corresponding locus in rats contains 28 such genes. One of these new genes has changed tissue specificity and has been found to be expressed primarily in vascular smooth muscle cells, and therefore been named rat vascular chymase (RVC). This β-chymase has been claimed to be a potent angiotensin-converting enzyme by cleaving angiotensin (Ang) I into Ang II and thereby having the potential to regulate blood pressure. To further characterize this enzyme, we have used substrate phage display and a panel of recombinant substrates to obtain a detailed quantitative view of its extended cleavage specificity. RVC was found to show a strong preference for Phe and Tyr in the P1 position, but also to accept Leu and Trp in this position. A strong preference for Ser or Arg in the P1’ position, just C-terminally of the cleavage site, and a preference for aliphatic amino acids in most other positions surrounding the cleavage site was also seen. Interesting also was a relatively strict preference for Gly in positions P3’ and P4’. RVC thereby shares similarity in its specificity to the mouse mucosal mast cell chymase mMCP-1, which efficiently converts Ang I to Ang II. This similarity adds support for the role of β-chymases as potent angiotensin converters in rodents, as their α-chymases, which have the capacity to efficiently convert Ang I into Ang II in other mammalian lineages, have become elastases. However, interestingly we found that RVC cleaved both after Arg2 and Phe8 in Ang I. Furthermore this cleavage was more than two hundred times less efficient than the consensus site obtained from the phage display analysis, indicating that RVC has a very low ability to cleave Ang I, raising serious doubts about its role in Ang I conversion.

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates.

The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1′, and P2′ variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1′ amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.

Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Effects of hydroxyl, carboxyl, and amino functionalized carbon nanotubes on the functional diversity of microbial community in riverine sediment

Nowadays, more and more attention is focused on the environmental harm brought by the wide production and use of carbon nanotubes. In this study, the metabolic function of sediment microbial community was investigated after unfunctionalized or functionalized multi-walled carbon nanotubes (MWCNTs) were incorporated. The surface functional groups on the studied functionalized MWCNTs in this work were hydroxyl, carboxyl, and amino, respectively. The metabolic functional diversity was determined by Biolog EcoPlates after one-month exposure to MWCNTs. Incorporating 0.5 wt% amino functionalized MWCNTs significantly decreased the microbial activity and diversity, and all types of MWCNTs caused great inhibition on the microbial metabolism at the dosage of 2.0 wt%. The sediment microbes preferred polymers and amino acids. Principal component and similarity analysis indicated that the microbial carbon metabolism was more affected by the MWCNT dosage compared with the functionalization, and 2.0 wt% amino functionalized MWCNTs made the greatest difference in metabolic function of sediment microbial community. These consequences may help to assess the environmental risks of MWCNTs from the aspect of ecological relevance of sediment microbial community.