scholarly journals Elucidation of the signatures of proteasome-catalysed peptide splicing

2020 ◽  
Author(s):  
Wayne Paes ◽  
German Leonov ◽  
Thomas Partridge ◽  
Annalisa Nicastri ◽  
Nicola Ternette ◽  
...  
Keyword(s):  
De Novo ◽  
Human Leukocyte ◽  
Amino Acid Residues ◽  
Peptide Fragments ◽  
Leukocyte Antigen ◽  
Peptide Length ◽  
Binding Pockets ◽  
Future Prediction ◽  
Endogenous Proteins

AbstractProteasomes catalyse the degradation of endogenous proteins into oligopeptides, but can concurrently create spliced oligopeptides through ligation of previously non-contiguous peptide fragments. Recent studies have uncovered a formerly unappreciated role for proteasome-catalysed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound cis-spliced peptides that can be targeted by CD8+ T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalysed cis-splicing reactions by examination of in vitro proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based de novo sequencing workflow. We show that forward and reverse PCPS reactions display unique splicing signatures, defined by preferential fusion of distinct amino acid residues with stringent peptide length distributions, suggesting sequence- and size-dependent accessibility of splice reactants for proteasomal substrate binding pockets. Our data provide the basis for a more informed mechanistic understanding of PCPS that will facilitate future prediction of spliced peptides from protein sequences.

scholarly journals Cross-Genotypic Examination of Hepatitis C Virus Polymerase Inhibitors Reveals a Novel Mechanism of Action for Thumb Binders

2014 ◽  
Vol 58 (12) ◽  
pp. 7215-7224 ◽  
Author(s):  
Auda A. Eltahla ◽  
Enoch Tay ◽  
Mark W. Douglas ◽  
Peter A. White
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Hcv Rna ◽  
Amino Acid Residues ◽  
Recombinant Enzymes ◽  
Polymerase Inhibitors ◽  
Binding Pockets ◽  
Direct Acting

ABSTRACTDirect-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well asin vitrotranscription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm β-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but notde novoactivity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced thede novoactivity at concentrations of ≥100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement ofde novoactivity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies.

scholarly journals Quality Verification with a Cluster−Controlled Manufacturing System to Generate Monocyte−Derived Dendritic Cells

Vaccines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 533
Author(s):  
Haruhiko Kawaguchi ◽  
Takuya Sakamoto ◽  
Terutsugu Koya ◽  
Misa Togi ◽  
Ippei Date ◽  
...  
Keyword(s):  
Cell Culture ◽  
Dna Microarrays ◽  
Cluster Formation ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Dc Vaccines ◽  
Survival Gene ◽  
Adherent Culture ◽  
Ctl Induction

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte−derived DCs induced by interleukin (IL)−4 with a low−adherence dish (low−adherent IL-4−DCs: la−IL-4−DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4−DCs developed using an adherent culture protocol. However, la−IL-4−DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la−IL-4−DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4−DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4−DCs. Mature IL-4−DCs in cell culture vessels (cluster−controlled IL-4−DCs: cc−IL-4−DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la−IL-4−DCs. cc−IL-4−DCs induced antigen−specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)−restricted melanoma antigen recognized by T cells 1 (MART−1) peptide. Additionally, cc−IL-4−DCs produced higher levels of IFN−γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro−survival gene. According to these findings, the cc−IL-4−DCs are useful for generating homogeneous and functional IL-4−DCs that would be expected to promote long−lasting effects in DC vaccines.

Human leukocyte antigen-DR negative de novo acute myeloid leukemia with Philadelphia chromosome

2008 ◽  
Vol 88 (5) ◽  
pp. 602-605
Author(s):  
Tohru Inaba ◽  
Hiroshi Nishimura ◽  
Junko Saito ◽  
Yoko Yamane ◽  
Takuya Nakatani ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Human Leukocyte Antigen ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Philadelphia Chromosome ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Acute Myeloid

scholarly journals Distinct Subregions of Swi1 Manifest Striking Differences in Prion Transmission and SWI/SNF Function

2010 ◽  
Vol 30 (19) ◽  
pp. 4644-4655 ◽  
Author(s):  
Zhiqiang Du ◽  
Emily T. Crow ◽  
Hyun Seok Kang ◽  
Liming Li
Keyword(s):  
Chromatin Remodeling ◽  
De Novo ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Conformational Switch ◽  
Amyloid Fibers ◽  
Aggregation Pattern ◽  
Prion Transmission ◽  
Shed Light

ABSTRACT We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI +], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for [SWI +] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI +] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in [SWI+ ] cells but diffuse in [swi− ] cells; chromosomal deletion of the N-coding region resulted in [SWI +] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI +] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in [SWI +] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of “protein-only” transmission of [SWI +] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

scholarly journals Human Herpesvirus 6A and 6B inhibit in vitro angiogenesis by induction of Human Leukocyte Antigen G

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Roberta Rizzo ◽  
Maria D’Accolti ◽  
Daria Bortolotti ◽  
Francesca Caccuri ◽  
Arnaldo Caruso ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Human Herpesvirus ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Human Leukocyte Antigen G ◽  
In Vitro Angiogenesis

scholarly journals Targeting human leukocyte antigen G with chimeric antigen receptors of natural killer cells convert immunosuppression to ablate solid tumors

2021 ◽  
Vol 9 (10) ◽  
pp. e003050
Author(s):  
Chia-Ing Jan ◽  
Shi-Wei Huang ◽  
Peter Canoll ◽  
Jeffrey N Bruce ◽  
Yu-Chuan Lin ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Natural Killer ◽  
Solid Tumors ◽  
Low Dose ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Low Dose Chemotherapy ◽  
Human Leukocyte Antigen G

BackgroundImmunotherapy against solid tumors has long been hampered by the development of immunosuppressive tumor microenvironment, and the lack of a specific tumor-associated antigen that could be targeted in different kinds of solid tumors. Human leukocyte antigen G (HLA-G) is an immune checkpoint protein (ICP) that is neoexpressed in most tumor cells as a way to evade immune attack and has been recently demonstrated as a useful target for chimeric antigen receptor (CAR)-T therapy of leukemia by in vitro studies. Here, we design and test for targeting HLA-G in solid tumors using a CAR strategy.MethodsWe developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification. A single-chain variable fragment (scFv) against HLA-G is designed as the targeting moiety, and the construct is tested both in vitro and in vivo on four different solid tumor models. We also evaluated the synergy of this anti-HLA-G CAR-NK strategy with low-dose chemotherapy as combination therapy.ResultsHLA-G CAR-transduced NK cells present effective cytolysis of breast, brain, pancreatic, and ovarian cancer cells in vitro, as well as reduced xenograft tumor growth with extended median survival in orthotopic mouse models. In tumor coculture assays, the anti-HLA-G scFv moiety promotes Syk/Zap70 activation of NK cells, suggesting reversal of the HLA-G-mediated immunosuppression and hence restoration of native NK cytolytic functions. Tumor expression of HLA-G can be further induced using low-dose chemotherapy, which when combined with anti-HLA-G CAR-NK results in extensive tumor ablation both in vitro and in vivo. This upregulation of tumor HLA-G involves inhibition of DNMT1 and demethylation of transporter associated with antigen processing 1 promoter.ConclusionsOur novel CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread. Further ablation of tumors can be boosted when combined with administration of chemotherapeutic agents in clinical use. The readiness of this novel strategy envisions a wide applicability in treating solid tumors.

scholarly journals An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

2019 ◽  
Author(s):  
Thomas F. Martinez ◽  
Qian Chu ◽  
Cynthia Donaldson ◽  
Dan Tan ◽  
Maxim N. Shokhirev ◽  
...  
Keyword(s):  
De Novo ◽  
Transcriptome Assembly ◽  
Human Leukocyte ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
Proteomics Data ◽  
Protein Coding ◽  
Leukocyte Antigen ◽  
Human Genes ◽  
Small Open Reading Frames

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

scholarly journals Immunosuppression with Calcineurin Inhibitor after Renal Transplant Failure Inhibits Allosensitization

Biomedicines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 72 ◽  
Author(s):  
Covadonga López del Moral Cuesta ◽  
Sandra Guiral Foz ◽  
David Gómez Pereda ◽  
José Luis Pérez Canga ◽  
Marina de Cos Gómez ◽  
...  
Keyword(s):  
Graft Failure ◽  
Calcineurin Inhibitor ◽  
De Novo ◽  
Immunosuppressive Treatment ◽  
Graft Loss ◽  
Multivariate Logistic Regression Analysis ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Single Antigen ◽  
Transplant Failure

Immunosuppression withdrawal after graft failure seems to favor sensitization. A high percentage of calculated panel-reactive antibody (cPRA) and the development of de novo donor specific antibodies (dnDSA) indicate human leukocyte antigen (HLA) sensitization and may hinder the option of retransplantation. There are no established protocols on the immunosuppressive treatment that should be maintained after transplant failure. A retrospective analysis including 77 patients who lost their first renal graft between 1 January 2006–31 December 2015 was performed. Two sera were selected per patient, one immediately prior to graft loss and another one after graft failure. cPRA was calculated by Single Antigen in all patients. It was possible to analyze the development of dnDSA in 73 patients. By multivariate logistic regression analysis, the absence of calcineurin inhibitor (CNI) at 6 months after graft failure was related to cPRA > 75% (OR 4.8, CI 95% 1.5–15.0, p = 0.006). The absence of calcineurin inhibitor (CNI) at 6 months after graft loss was significantly associated with dnDSA development (OR 23.2, CI 95% 5.3–100.6, p < 0.001). Our results suggest that the absence of CNI at the sixth month after graft loss is a risk factor for sensitization. Therefore, maintenance of an immunosuppressive regimen based on CNI after transplant failure should be considered when a new transplant is planned, since it seems to prevent HLA allosensitization.

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