Protein Sequence Variation Research Articles

Undesirable protein sequence variations in maize genes that confer resistance to fungal pathogens and insect pests

Diseases and insect pests greatly impact sustainable production in maize. Maize inbred lines have varying levels of resistance to these pathogens and insects, but little is known about the diversity of their resistance proteins. In this study, genes encoding seven proteins that are involved in resistance to insects and pathogens in maize were analyzed in 46 maize inbred lines to elucidate the differences in amino acid sequences. The proteins of interest are superlectin, maizewin, hydrolase, geranyl geranyl transferase, quinone oxidoreductase, AIL1, and defensin. The protein sequences encoded by genes for superlectin, AIL1, and defensin were found to be disrupted in some maize inbreds but were conserved in others. The characterized disruptions resulted from single amino acid changes, insertions, or deletions. While the effect of single amino acid changes is hard to predict, insertions and deletions likely disrupt protein function, increasing the susceptibility of maize plants to insects and/or diseases. Functional resistance genes can be incorporated from the identified maize inbreds into commercial hybrids to promote enhanced insect and pathogen resistance.

GENERALIST: A latent space based generative model for protein sequence families.

Generative models of protein sequence families are an important tool in the repertoire of protein scientists and engineers alike. However, state-of-the-art generative approaches face inference, accuracy, and overfitting- related obstacles when modeling moderately sized to large proteins and/or protein families with low sequence coverage. Here, we present a simple to learn, tunable, and accurate generative model, GENERALIST: GENERAtive nonLInear tenSor-factorizaTion for protein sequences. GENERALIST accurately captures several high order summary statistics of amino acid covariation. GENERALIST also predicts conservative local optimal sequences which are likely to fold in stable 3D structure. Importantly, unlike current methods, the density of sequences in GENERALIST-modeled sequence ensembles closely resembles the corresponding natural ensembles. Finally, GENERALIST embeds protein sequences in an informative latent space. GENERALIST will be an important tool to study protein sequence variability.

Genetic preservation of SLC22A3 in the Admixed and Xhosa populations living in the Western Cape

BackgroundAmphiphilic solute facilitator organic cation transporters mediate the movement of various endogenous and exogenous organic cations, including crucial drugs like metformin, oxaliplatin, and lamivudine. These transporters are now seen as a potential explanation for inter-individual differences in drug effectiveness, contributing to 15–30% of such variability due to genetic factors.The aim of this study was to determine the baseline minor allele frequency distribution of 18 known coding SNPs in the SLC22A3 gene of 278 Cape Admixed (130) and Xhosa (148) individuals residing in Cape Town, South Africa.MethodsA convenience sampling method was used for sample collection. DNA extraction and subsequent amplification of target sites was carried out according to standard established methodologies. All genotyping was performed using the SNaPshot™ mini-seuqencing platform.ResultsThis study found no genetic polymorphisms in the coding region of the SLC22A3 gene of both the Xhosa and Cape Admixed individuals investigated.ConclusionThis study has shown that SLC22A3 coding SNPs observed in other populations are absent in the sample of both Cape Admixed and Xhosa individuals studied. The lack of protein sequence variation was consistent with other studies and may reflect the significant physiological role of human organic cation transporter 3 in maintaining cellular and organismal homeostasis.

A Catalog of Coding Sequence Variations in Salivary Proteins' Genes Occurring during Recent Human Evolution.

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.

Application of deep mutational scanning technology in protein research

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.

Amino Acid Variation at the Mitochondrial Binding Site of Antimycin A is Proposed to Reflect Sensitivity and Toxicity Differences among Fish Species

To better understand differential sensitivities among fish species to the piscicidal compound Antimycin-A (ANT-A), we hypothesized that variations in amino acids at the ANT-A binding site may reflect toxicity differences. Protein sequences for six motifs comprising the ANT-A binding site were obtained and compared for invasive carp species (N = 515) and seven non-target species (N = 277); a consensus was delineated from each species. The carp species, Common Carp (Cyprinus carpio), Silver Carp (Hypophthalmichthys molitrix), Bighead Carp (Hypophthalmichthys nobilis), Grass Carp (Ctenopharyngodon idella), and Black Carp (Mylopharyngodon piceus), showed the same amino acids at the site; thus, it was termed the carp consensus motif sequence (CCM). Channel Catfish (Ictalurus punctatus) showed the most amino acid polymorphisms, with three motifs 96–100% different from CCM. Within a species, Bluegill (Lepomis macrochirus) and Fathead Minnow (Pimephales promelas) variation per motif was most dissimilar (46.7% and 21.6%, respectively). Organismal mortality data from the literature indicated Yellow Perch (Perca flavescens), Walleye (Sander vitreus), and American Gizzard Shad (Dorosoma cepedianum) to be most sensitive to the piscicide, Catfish least sensitive, and all others intermediate. The protein sequence variations of the binding site appeared to be in accord with organismal sensitivity categories when they differed from the CCM; the motifs in Gizzard Shad and Walleye were the same as in CCM. The physical/chemical nature of ANT-A is important to consider in organismal response comparisons. This cellular approach of studying ANT-A binding at its target enzyme is a non-destructive way to predict piscicidal efficacy of ANT-A against fishes of interest, informs management decisions in control efforts for invasives, and can be used to forecast effects on sympatric species.

Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Evolutionary Relationship and Structural Analysis of Blast Resistance Associated Novel Osvwa36 and Osvwa37 Genes in Cultivated and Wild Species of Rice

Rice blast is a dreadful disease that causes enormous losses in rice production worldwide. To develop blast resistant rice cultivars, it is necessary to identify resistance and defence regulator genes and the underlying mechanism of resistance. A novel von Willebrand factor domain A containing genes OsvWA36 and OsvWA37 in Tetep cultivar of rice regulate response to Magnaporthe oryzae infection and provides significant resistance. Owing to the important role of these genes, their evolutionary relationship has been studied in cultivated and wild species of rice. There is significant diversity in the protein sequence of these genes among the relative wild rice species. The size of OsvWA36 protein varies from 501 aa to 698 aa whereas size of OsvWA37 protein varies from 295 aa to 1004 aa. The, OsvWA36 gene is evolutionarily more conserved than OsvWA37 among the different rice species indicating its critical role. Besides the global variation in protein sequence, the region of vWA domain is highly conserved among all the species. Interestingly, both the genes in Oryza barthii are fused to form a single gene encoding a large protein that indicates their origin in other species from a single gene. The good quality tertiary structures of both OsvWA36 and OsvWA37 proteins in cultivated germplasm cv. Tetep were also generated which can be utilized for protein structural and docking studies.

Sequence-dependent model of allosteric communication.

The omnipresence and diversity of allosteric regulation in proteins and protein associations complemented by the potential for the design of allosterically acting biologics and drugs call for the development of a new generation of computational models for the analysis of allostery and rational engineering/design of desired signaling and effector molecules determining it. One of the most important challenges is the consideration of the role of amino acid sequence in forming the protein's allosteric communication, including the mode and strength of the allosteric signal that is communicated to the regulated functional site. Here, we present the network-based model with a sequence dependence added in consideration of allosteric communication by combining the structure-based statistical mechanical model of allostery with the Miyazawa-Jernigan residue-residue potential. Applying the model in the analysis of five classical allosteric proteins, we found that it is necessary to consider the following two major determinants: (i) the free energy exerted by the allosteric site on the regulated one and (ii) the background (average) change in dynamics of the overall structure. We show that working together these two components determine the allosteric modulation, calling one to study their dependence on structures, oligomerization states, and sequence divergence in different proteins.

HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review

HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47–59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47–59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.

Evolutionary trend of bovine β-defensin proteins toward functionality prediction: A domain-based bioinformatics study

Defensins are small cationic cysteine-rich and amphipathic peptides that form of three-dimensional β-strand structure connected by disulfide bonds. Defensins form key elements of the innate immune system of multicellular organisms. They not only possess broad-spectrum antimicrobial activity but also have diverse roles, including cell signaling, ion channel agitation, toxic functions, and enzyme inhibitor activities in various animals. Although the role of β-defensins in immune responses against infectious agents and reproduction could be significant, inadequate genomic information is available to explain the whole β-defensin repertoire in cattle. No domain or motif-based functional analyses have been previously reported. In addition, how do defensins possess this magnitude of functions in the immune system is still not clear. Our present study, therefore, investigated the sequence divergence and evolutionary relations of bovine defensin proteins with those of humans. Our domain-based evolutionary analysis revealed four major clusters with significant domain variation while reserving a main antimicrobial activity. Our study revealed the β-defensin domain as the ancestor domain, and it is preserved in the first group of defensin protein with no α-helix in its structure. Due to natural selection, some domains have evolved independently within clusters II and III, while some proteins have lost their domain characteristics. Cluster IV contains the most recently evolved domains. Some proteins of all but cluster I might have adopted the functional characteristics of α-defensins which is largely absent in cattle. The proteins show different patterns of disulfide bridges and multiple signature patterns which might render them specialized functions in different tissue to combat against various pathogens.

Using New Peak Detection to Solve Sequence Variants Analysis Challenges in Bioprocess Development.

Recombinant therapeutic proteins have become the major class of drugs to treat various human diseases in recent years. Low levels of protein sequence variants (SVs) have been reported to be present in recombinant therapeutic proteins. The consequences of potential unwanted immune response from SVs of recombinant therapeutic proteins have increasingly drawn attention from regulatory authorities and the biopharmaceutical industry. It is highly desirable to detect low-level SVs during clone selection and early process development as part of the control strategy. Peptide mapping with LC-MS/MS analysis has been applied as a powerful tool to characterize post-translation modifications of therapeutic proteins. Despite the recent advancements in mass spectrometry hardware and software, it is still quite challenging and time-consuming to detect and identify low-level SVs. In this study, we present an optimized approach using new peak detection to detect and identify low level SVs with high confidence and high speed. The new approach makes sequence variants analysis by LC-MS/MS broadly applicable and practical in bioprocess development of therapeutic proteins.

Two haplotype-resolved, gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit

Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value, especially because of the high content of vitamin C in its fruit. In this study, we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var. ‘Donghong’ (DH) and Actinidia latifolia ‘Kuoye’ (KY), with total lengths of 608 327 852 and 640 561 626 bp for 29 chromosomes, respectively. With a burst of structural variants involving inversion, translocations, and duplications within 8.39 million years, the metabolite content of DH and KY exhibited differences in saccharides, lignans, and vitamins. A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha, both of which have ultra-high vitamin C content. With each assembly phased into two complete haplotypes, we identified allelic variations between two sets of haplotypes, leading to protein sequence variations in 26 494 and 27 773 gene loci and allele-specific expression of 4687 and 12 238 homozygous gene pairs. Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents; free fatty acids and flavonols accumulated in the early stages, but sugar substances and amino acids accumulated in the late stages. The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit. Compared with wild varieties and other Actinidia species, AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content, providing a possible explanation on why red-flesh kiwifruits are sweeter. Collectively, these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.

Group A Streptococcus Infections: Their Mechanisms, Epidemiology, and Current Scope of Vaccines.

Group A streptococci (GAS) are gram-positive, cocci-shaped bacteria that cause a wide variety of infections and are a cause of significant health burden, particularly in lower- and middle-income nations. The GAS genome contains a number of virulence factors such as the M-protein, hyaluronic acid, C5a peptidase, etc. Despite its significant health burden across the globe, a proper vaccine against GAS infections is not yet available. Various candidates for an effective GAS vaccine are currently being researched. These are based on various parts of the streptococcal genome. These include candidates based on the N-terminal region of the M protein, the conserved C-terminal region of the M protein, and other parts of the streptococcal genome. The development of a vaccine against GAS infections is hampered by certain challenges, such as extensive genetic heterogeneity and high protein sequence variation. This review paper sheds light on the various virulence factors of GAS, their epidemiology, the different vaccine candidates currently being researched, and the challenges associated with M-protein and non-M-protein-based vaccines. This review also sheds light on the current scenario regarding the status of vaccine development against GAS-related infections.

Genetic and functional odorant receptor variation in the Homo lineage

Humans, Neanderthals, and Denisovans independently adapted to a wide range of geographic environments and their associated food odors. Using ancient DNA sequences, we explored the invitro function of thirty odorant receptor genes in the genus Homo. Our extinct relatives had highly conserved olfactory receptor sequence, but humans did not. Variations in odorant receptor protein sequence and structure may have produced variation in odor detection and perception. Variants led to minimal changes in specificity but had more influence on functional sensitivity. The few Neanderthal variants disturbed function, whereas Denisovan variants increased sensitivity to sweet and sulfur odors. Geographic adaptations may have produced greater functional variation in our lineage, increasing our olfactory repertoire and expanding our adaptive capacity. Our survey of olfactory genes and odorant receptors suggests that our genus has a shared repertoire with possible local ecological adaptations.

94. Pooled analysis of nirsevimab resistance through 150 days post dose in preterm and term infants

Abstract Background Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection (LRTI) and hospitalization in infants. In two global pivotal placebo-controlled studies, nirsevimab, a monoclonal antibody to the RSV prefusion (F) protein with extended half-life, reduced medically attended (MA) RSV LRTI versus placebo throughout the RSV season (MELODY (Primary Cohort)/Study 3 (Proposed Dose) Pool, 79.5% efficacy). Here we summarize resistance analyses of all RT-PCR-confirmed RSV isolates from healthy term and preterm infants through 150 days post dose. Methods Infants were randomized 2:1 to receive one intramuscular injection of nirsevimab or placebo, prior to their first RSV season. RT-PCR-confirmed RSV isolates were reflexed for genotypic analyses of RSV F and phenotypic analyses of identified substitutions in a recombinant RSV neutralization susceptibility assay. Results In the pooled proposed dose analysis of Study 3 (50 mg nirsevimab if < 5 kg at dosing) and MELODY (50 or 100 mg nirsevimab if < 5 kg or ≥5 kg at dosing, respectively), no subject with MA RSV LRTI had an RSV isolate containing nirsevimab resistance-associated substitutions in either treatment group (nirsevimab, RSV A: 0/14 and RSV B: 0/5; placebo, RSV A: 0/35 and RSV B: 0/16). In Study 3 (50 mg nirsevimab if ≥5 kg at dosing), 2/18 subjects in the nirsevimab group and 0/20 subjects in the placebo group with MA RSV LRTI had an RSV isolate harbouring nirsevimab binding site substitutions I64T+K68E+I206M+Q209R (>447-fold) or N208S (>387-fold) that conferred reduced susceptibility to nirsevimab neutralization (nirsevimab, RSV A: 0/9 and RSV B: 2/9; placebo, RSV A: 0/10 and RSV B: 0/10). Subjects with RSV isolates harboring F protein sequence variations that maintained susceptibility to nirsevimab neutralization were balanced between treatment groups with no association with RSV disease severity. No subjects with non-protocol defined MA RSV LRTI cases or hospitalization due to any RSV respiratory illness had an RSV isolate conferring nirsevimab resistance. Conclusion Lack of nirsevimab resistance following immunization at the proposed dose supports efficacy and neutralization activity of nirsevimab against both RSV A and B strains throughout the RSV season. Disclosures Michael E. Abram, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Bahar Ahani, BSC, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds David E. Tabor, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Fiona Fernandes, PhD, AstraZeneca: Stocks/Bonds Deidre Wilkins, BSC, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Anastasia A. Aksyuk, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Kevin M. Tuffy, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Hong Ji, BSc, AstraZeneca: Stocks/Bonds Christine Blaze, BSc, AstraZeneca: Stocks/Bonds Tyler Brady, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Pamela Griffin, MD, AstraZeneca: Stocks/Bonds Amanda Leach, MD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Tonya L. Villafana, PhD, MPH, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Stocks/Bonds.

Identifying individuals using proteomics: are we there yet?

Multi-omics approaches including proteomics analyses are becoming an integral component of precision medicine. As clinical proteomics studies gain momentum and their sensitivity increases, research on identifying individuals based on their proteomics data is here examined for risks and ethics-related issues. A great deal of work has already been done on this topic for DNA/RNA sequencing data, but it has yet to be widely studied in other omics fields. The current state-of-the-art for the identification of individuals based solely on proteomics data is explained. Protein sequence variation analysis approaches are covered in more detail, including the available analysis workflows and their limitations. We also outline some previous forensic and omics proteomics studies that are relevant for the identification of individuals. Following that, we discuss the risks of patient reidentification using other proteomics data types such as protein expression abundance and post-translational modification (PTM) profiles. In light of the potential identification of individuals through proteomics data, possible legal and ethical implications are becoming increasingly important in the field.

Genetic analysis of a 66-kDa protein-encoding gene of Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

The rat lungworm Angiostrongylus cantonensis is globally known to be the cause of oeosinophilic meningitis in humans. Another congener, Angiostrongylus malaysiensis, is closely related to A. cantonensis and has been described as a potential human pathogenic parasite. These 2 worms are similar in terms of life cycle, host range and morphological and genetic information. However, there are limited studies about their genetic diversity based on the 66-kDa protein-encoding gene. The objective of this study was to explore the 66-kDa protein sequence variation of A. cantonensis and A. malaysiensis collected from Thailand. Two adult and 53 third-stage larval specimens of Angiostrongylus from 4 geographic locations in Thailand were molecularly identified using the 66-kDa protein gene. The phylogenetic trees (Bayesian inference tree and maximum-likelihood tree) showed that Angiostrongylus formed a monophyletic clade with a clear separation between A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis varies from 0.82 to 2.86%, with a total of 16 variable sites. The analysis of genetic diversity revealed 1 and 5 new haplotypes of A. cantonensis and A. malaysiensis, respectively, and showed genetic differences between the populations of A. cantonensis and A. malaysiensis. The haplotype networks of A. cantonensis and A. malaysiensis populations in Thailand are similar to those of populations in some countries, indicating the range expansion of genomic origin between populations in different areas. In conclusion, the 66-kDa protein gene was a good genetic marker for studying genetic diversity and discriminating between A. cantonensis and A. malaysiensis.

Host KIR/HLA-C Genotypes Determine HIV-Mediated Changes of the NK Cell Repertoire and Are Associated With Vpu Sequence Variations Impacting Downmodulation of HLA-C.

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1+ individuals. Compared to 60 HIV-1- controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C+ and KIR3DL2+ NK cell sub-populations from HIV-1+ individuals was enlarged compared to HIV-1- controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1+ NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1.

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase-zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody-drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes.