scholarly journals ARTEMIS: A Novel Mass-Spec Platform for HLA-Restricted Self and Disease-Associated Peptide Discovery

2021 ◽  
Vol 12 ◽  
Author(s):  
Kathryn A. K. Finton ◽  
Mi-Youn Brusniak ◽  
Lisa A. Jones ◽  
Chenwei Lin ◽  
Andrew J. Fioré-Gartland ◽  
...  
Keyword(s):  
Hek293 Cells ◽  
Middle Ground ◽  
Affinity Tag ◽  
Hla Antibodies ◽  
Multiple Alleles ◽  
Lentiviral Transduction ◽  
Mass Spec ◽  
Allele Specific ◽  
Specific Proteins ◽  
Computational Predictions

Conventional immunoprecipitation/mass spectroscopy identification of HLA-restricted peptides remains the purview of specializing laboratories, due to the complexity of the methodology, and requires computational post-analysis to assign peptides to individual alleles when using pan-HLA antibodies. We have addressed these limitations with ARTEMIS: a simple, robust, and flexible platform for peptide discovery across ligandomes, optionally including specific proteins-of-interest, that combines novel, secreted HLA-I discovery reagents spanning multiple alleles, optimized lentiviral transduction, and streamlined affinity-tag purification to improve upon conventional methods. This platform fills a middle ground between existing techniques: sensitive and adaptable, but easy and affordable enough to be widely employed by general laboratories. We used ARTEMIS to catalog allele-specific ligandomes from HEK293 cells for seven classical HLA alleles and compared results across replicates, against computational predictions, and against high-quality conventional datasets. We also applied ARTEMIS to identify potentially useful, novel HLA-restricted peptide targets from oncovirus oncoproteins and tumor-associated antigens.

Incidence and impact of allele-specific anti-HLA antibodies and high-resolution HLA genotyping on assessing immunologic compatibility

2021 ◽  
Vol 82 (3) ◽  
pp. 147-154
Author(s):  
Daria Zavyalova ◽  
Joseph Abraha ◽  
Ping Rao ◽  
Gerald P. Morris
Keyword(s):  
High Resolution ◽  
Hla Antibodies ◽  
Hla Genotyping ◽  
Allele Specific

scholarly journals Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 650 ◽  
Author(s):  
Rachel M. Carnes ◽  
Robert A. Kesterson ◽  
Bruce R. Korf ◽  
James A. Mobley ◽  
Deeann Wallis
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Affinity Purification ◽  
Neurofibromatosis Type ◽  
Hek293 Cells ◽  
Ras Signaling ◽  
Affinity Tag ◽  
Pathogenic Variants ◽  
Binding Partners ◽  
Keratin Expression

Neurofibromatosis Type 1 (NF1) is caused by pathogenic variants in the NF1 gene encoding neurofibromin. Definition of NF1 protein–protein interactions (PPIs) has been difficult and lacks replication, making it challenging to define binding partners that modulate its function. We created a novel tandem affinity purification (TAP) tag cloned in frame to the 3’ end of the full-length murine Nf1 cDNA (mNf1). We show that this cDNA is functional and expresses neurofibromin, His-Tag, and can correct p-ERK/ERK ratios in NF1 null HEK293 cells. We used this affinity tag to purify binding partners with Strep-Tactin®XT beads and subsequently, identified them via mass spectrometry (MS). We found the tagged mNf1 can affinity purify human neurofibromin and vice versa, indicating that neurofibromin oligomerizes. We identify 21 additional proteins with high confidence of interaction with neurofibromin. After Metacore network analysis of these 21 proteins, eight appear within the same network, primarily keratins regulated by estrogen receptors. Previously, we have shown that neurofibromin levels negatively regulate keratin expression. Here, we show through pharmacological inhibition that this is independent of Ras signaling, as the inhibitors, selumetinib and rapamycin, do not alter keratin expression. Further characterization of neurofibromin oligomerization and binding partners could aid in discovering new neurofibromin functions outside of Ras regulation, leading to novel drug targets.

scholarly journals S49G and R389G polymorphisms of the β1-adrenergic receptor influence signaling via the cAMP-PKA and ERK pathways

2013 ◽  
Vol 45 (23) ◽  
pp. 1186-1192 ◽  
Author(s):  
Fan Zhang ◽  
Susan F. Steinberg
Keyword(s):  
Heart Failure ◽  
Lipid Raft ◽  
Adrenergic Receptor ◽  
Hek293 Cells ◽  
Blocker Therapy ◽  
Erk Activation ◽  
Camp Pathway ◽  
Biochemical Fractionation ◽  
Allele Specific ◽  
Β Blocker

Two functionally important β1-adrenergic receptor (β1AR) polymorphisms have been identified. The R389G polymorphism influences coupling to the Gs-cAMP pathway. R389-β1ARs display enhanced activation of cAMP/PKA; they provide short-term inotropic support but also cause a predisposition to cardiomyopathic decompensation. A second S49G polymorphism is implicated in the evolution of heart failure, but the mechanism remains uncertain. This study shows that position 49 and 389 polymorphisms function in a coordinate manner to influence agonist-dependent cAMP/PKA and ERK responses. cAMP/PKA and ERK responses are more robust in HEK293 cells that heterologously overexpress G49-β1ARs, compared with S49-β1ARs. However, this phenotype is most obvious on a G389-β1AR background; the more robust agonist-dependent cAMP/PKA and ERK responses in R389-β1AR cells effectively obscure the effect of the S49G polymorphism. We also show that isoproterenol (Iso) and carvedilol activate ERK via a similar EGFR-independent mechanism in cells expressing various β1AR haplotypes. However, Iso activates ERK via an Src-independent pathway, but carvedilol-dependent ERK activation requires Src. Since the S49G polymorphism has been linked to changes in β1AR trafficking, we examined whether β1AR polymorphisms influence partitioning to lipid raft membranes. Biochemical fractionation studies show that all four β1AR variants are recovered in buoyant flotillin-enriched membranes; the distinct signaling phenotypes of the different β1AR variants could not be attributed to any gross differences in basal compartmentalization to lipid raft membranes. The allele-specific differences in β1AR signaling phenotypes identified in this study could underlie interindividual differences in responsiveness to β-blocker therapy and clinical outcome in heart failure.

scholarly journals Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies potential targets of allele-specific immunity to clinical malaria

2020 ◽  
Author(s):  
Zalak Shah ◽  
Myo T Naung ◽  
Kara A Moser ◽  
Matthew Adams ◽  
Andrea G Buchwald ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Data ◽  
Clinical Malaria ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Multiple Alleles ◽  
Vaccine Candidates ◽  
Genome Wide ◽  
Allele Specific

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. This immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode likely targets of naturally acquired immunity and that should be further characterized for their potential as vaccine candidates.

scholarly journals A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB–SPATA22 complex†

2020 ◽  
Vol 103 (2) ◽  
pp. 333-342 ◽  
Author(s):  
Yang Xu ◽  
Rong Liu ◽  
N Adrian Leu ◽  
Lei Zhang ◽  
Ilsiya Ibragmova ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Bifc Assay ◽  
Small Molecule Libraries ◽  
A Cell ◽  
Specific Proteins

Abstract MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins.

scholarly journals Generation of Unexpected Allele-Specific Anti-HLA Antibodies after the Transplantation of a Fully-Matched Kidney Allograft and the Diagnostic Approaches Required for Excluding Harmful Effects after Subsequent Renal Regrafts

2021 ◽  
Vol 05 (02) ◽  
pp. 1-1
Author(s):  
Gerald Schlaf ◽  
◽  
Jakob Kehlen ◽  
Anja Wahle ◽  
Diana Mauer ◽  
...  
Keyword(s):  
A Priori ◽  
Hla Antigens ◽  
Human Leukocyte ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hla Antibodies ◽  
Allele Specific ◽  
Diagnostic Approaches ◽  
Allelic Group ◽  
Class Ii Antigen

The specification of anti-human leukocyte antigen (HLA) antibodies is an important task for patients awaiting kidney allografts. Especially the patients immunized in previous transplantations, transfusions, or pregnancies must be carefully observed, since grafting patients with HLA antigens/phenotypes recognized by their pre-formed antibodies are the main cause of harmful hyperacute and acute rejection. The complement-dependent lymphocytotoxicity-based de facto (physical) crossmatching (CDC-CM) has thus been implemented as the last diagnostic obstacle before kidney allografting. Here, an assay is performed by incubating the donors’ lymphocytes with the sera of the prospective recipients, and a negative outcome was desired for eligibility of the underlying organ allocation. Furthermore, valid antibody specification has to be performed at least quarterly for each patient on the kidney waiting list, as defined by certain guidelines, for example, the Eurotransplant guidelines. Based on the exclusion of these specificities, also referred to as virtual crossmatching, certain donors are a priori listed as unacceptable for these recipients. In this case report, we showed that defining unacceptable antigens may be difficult if the recipients’ antibodies are allele-specific after being generated in the patient who is expressing the HLA-class II antigen DQ6 and also developing antibodies against this antigen. Low resolution (two-digit) typing is used before kidney allografting. Thus, these antibodies are generally not definable, as donors and recipients share the same antigen (allelic group). Here, we demonstrate the diagnostic approaches required to exclude inadequate kidney donors for a patient exhibiting antibodies only against the HLA-DQB1*06:04 allelic variant and not against the common phenotype HLA-DQ6. In practice, the patient’s HLA-class II high resolution (four-digit) typing, as well as his antibody specification at the highest (single antigen) resolution, are included. Furthermore, we critically discuss, according to the Eurotransplant guidelines, the missing possibility to declare own HLA-antigens unacceptable, which may be very helpful for recipients who exhibit allele-specific antibodies.

scholarly journals ISOLATION AND GENETIC ANALYSIS OF MUTATIONS AT THE SerH IMMOBILIZATION ANTIGEN LOCUS OF TETRAHYMENA THERMOPHILA

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 273-286
Author(s):  
F P Doerder ◽  
M S Berkowitz ◽  
J Skalican-Crowe
Keyword(s):  
Tetrahymena Thermophila ◽  
Surface Protein ◽  
Wild Type ◽  
Multiple Alleles ◽  
Immobilization Antigen ◽  
F1 Progeny ◽  
Allele Specific ◽  
Heterozygous Form ◽  
Interallelic Complementation ◽  
Type Strains

ABSTRACT Multiple alleles at the SerH locus specify the major cell surface protein (immobilization antigen) of the ciliate Tetrahymena thermophila. Following mutagenesis of SerH1 homozygotes, two mutations, H1-1 and H1-2, were recovered in heterozygous form. Mutant homozygotes do not express H1 antigen, nor is H1 expressed in F1 progeny of crosses to wild-type strains homozygous for SerH2 or SerH3. H1-1 and H1-2 segregate without recombination from these wild-type alleles in expected F2 and testcross Mendelian ratios. H1-1 and H1-2 do, however, complement each other to express H1 antigen. Experiments suggest this complementation is due neither to recombination during macronuclear development nor to interallelic complementation of defective SerH1 gene products. These results suggest that SerH1 is intact in one mutant, and possibly both, although no such allele has been segregated in testcross progeny (N = 205). The hypothesis is presented that complementation between H1-1 and H1-2 is due to interaction between allele-specific regulators closely linked to the SerH1 gene.

scholarly journals Quantitative resistance to Phytophthora infestans in potato: a case study for QTL mapping in an allogamous plant species.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 67-77 ◽  
Author(s):  
C Leonards-Schippers ◽  
W Gieffers ◽  
R Schäfer-Pregl ◽  
E Ritter ◽  
S J Knapp ◽  
...  
Keyword(s):  
Qtl Mapping ◽  
Phytophthora Infestans ◽  
Specific Resistance ◽  
Quantitative Resistance ◽  
Potato Virus X ◽  
Interval Mapping ◽  
Wild Potato Species ◽  
Resistance Response ◽  
Multiple Alleles ◽  
Specific Proteins

Abstract Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance--the latter being more important for potato breeding--are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F1 progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.

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