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Nature Communications Jun 2023Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains...
Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.
Topics: Humans; Phosphopeptides; Receptors, Antigen, T-Cell; Protein Binding
PubMed: 37353482
DOI: 10.1038/s41467-023-39425-1 -
Journal of Experimental & Clinical... Dec 2023Cancer cells undergo cellular adaptation through metabolic reprogramming to sustain survival and rapid growth under various stress conditions. However, how brain tumors...
BACKGROUND
Cancer cells undergo cellular adaptation through metabolic reprogramming to sustain survival and rapid growth under various stress conditions. However, how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment remain unknown.
METHODS
We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify the regulatory mechanisms of S/G deprivation-induced metabolic flexibility, we employed high-throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens.
RESULTS
The high-throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK-dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes the expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, the HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP ratio, and the proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that the levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and the poor prognosis of GBM patients.
CONCLUSION
Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells, and highlight the potential to target SSP for treating human GBM.
Topics: Humans; AMP-Activated Protein Kinases; Glioblastoma; Serine; Glucose; Glycine; RNA; Hypoxia-Inducible Factor 1, alpha Subunit; Cell Line, Tumor; Tumor Microenvironment; Phosphofructokinase-2
PubMed: 38098117
DOI: 10.1186/s13046-023-02927-3 -
MBio Aug 2023HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK...
HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.
Topics: Humans; Down-Regulation; HIV-1; Ligands; Antibody-Dependent Cell Cytotoxicity; Killer Cells, Natural; Signaling Lymphocytic Activation Molecule Family; HIV Infections
PubMed: 37404017
DOI: 10.1128/mbio.00789-23 -
Frontiers in Molecular Biosciences 2023This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory...
This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory cytokines and lipid subspecies and improved clinical outcomes in adults treated with an adjunctive ketogenic diet for super-refractory status epilepticus (SRSE). Adults (18 years or older) were treated with a 4:1 (fat: carbohydrate and protein) ratio of enteral KD as adjunctive therapy to pharmacologic seizure suppression in SRSE. Blood and urine samples and clinical measurements were collected at baseline ( = 10), after 1 week ( = 8), and after 2 weeks of KD ( = 5). In addition, urine acetoacetate, serum -hydroxybutyrate, lipidomics, pro-inflammatory cytokines (IL-1β and IL-6), chemokines (CCL3, CCL4, and CXCL13), and clinical measurements were obtained at these three time points. Univariate and multivariate data analyses were performed to determine the correlation between ketone body production and circulating lipids, inflammatory biomarkers, and clinical outcomes. Changes in lipids included an increase in ceramides, mono-hexosylceramide, sphingomyelin, phosphocholine, and phosphoserines, and there was a significant reduction in pro-inflammatory mediators, IL-6 and CXCL13, seen at 1 and 2 weeks of KD. Higher blood -hydroxybutyrate levels at baseline correlated with better clinical outcomes; however, ketone body production did not correlate with other variables during treatment. Higher chemokine CCL3 levels following treatment correlated with a longer stay in the intensive care unit and a higher modified Rankin Scale score (worse neurologic disability) at discharge and 6-month follow up. Adults receiving an adjunctive enteral ketogenic diet for super-refractory status epilepticus exhibit alterations in select pro-inflammatory cytokines and lipid species that may predict their response to treatment.
PubMed: 37936721
DOI: 10.3389/fmolb.2023.1173039 -
Nature Communications Nov 2023Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for...
Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-T cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM B) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.
Topics: Animals; Immunity, Humoral; Germinal Center; Adjuvants, Immunologic; Antigens; Primates; Antibodies, Neutralizing; HIV Infections; HIV Antibodies; env Gene Products, Human Immunodeficiency Virus
PubMed: 37925510
DOI: 10.1038/s41467-023-42923-x -
The Journal of Biological Chemistry Nov 2023Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18...
Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1 interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition. We found that SLF1 utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.
Topics: Protein Binding; Protein Domains; DNA Damage; Ubiquitin-Protein Ligases; Peptides; DNA Repair
PubMed: 37748650
DOI: 10.1016/j.jbc.2023.105288 -
NPJ Vaccines Aug 2023In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and...
In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.
PubMed: 37573422
DOI: 10.1038/s41541-023-00711-0 -
Acta Neuropathologica Communications Jul 2023Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis...
Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1β, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.
Topics: Animals; Mice; Antibodies, Monoclonal; Epitopes; Immunization; Intermediate Filaments; NF-kappa B
PubMed: 37434215
DOI: 10.1186/s40478-023-01592-z -
Phytomedicine : International Journal... Jul 2023Erchen decoction, a traditional Chinese medicine formula, can reduce the level of oxidative stress for the treatment of dyslipidemia phlegm-dampness retention syndrome...
Erchen decoction to reduce oxidative stress in dyslipidemia phlegm-dampness retention syndrome mice: In vivo mechanism revealed by metabolomics (liquid chromatography-mass spectrometry).
OBJECTIVE
Erchen decoction, a traditional Chinese medicine formula, can reduce the level of oxidative stress for the treatment of dyslipidemia phlegm-dampness retention syndrome (DPDRS); however, studies have not elucidated the mechanism underlying its metabolic action. Here, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic techniques were utilized to characterize the in vivo effects of Erchen decoction in achieving reduction of oxidative stress levels and understand the potential metabolic mechanisms of action.
METHODS
We constructed a DPDRS animal model using a multifactorial composite modeling approach, and Erchen decoction was administered by gavage. We employed LC-MS-based metabolomic techniques in combination with serum-associated factors, gene transcription, methylation detection, and hematoxylin and eosin staining.
RESULTS
In this study, the constructed animal model of DPDRS had satisfactory quality. Erchen decoction treatment reduced the levels of low-density lipoprotein cholesterol, t total cholesterol and riglyceride; it improved the endothelial structure, increased levels of serum β-nicotinamide adenine dinucleotide phosphate and glutathione concentrations, increased aortic phosphoserine aminotransferase and phosphoserine phosphatase gene expression levels, and decreased aortic phosphoglycerate dehydrogenase methylation level. A total of 64 differential metabolites were obtained using LC-MS assay, and 34 differential metabolic pathways were obtained after enrichment.
CONCLUSIONS
Erchen decoction treatment of DPDRS mice reversed lipid indexes, improved vascular endothelial structure, increased serum and aortic anti-oxidative stress factor concentration and expression levels, and decreased methylation levels, thereby reducing oxidative stress and protecting vascular endothelium. Tricarboxylic acid cycle and metabolic pathways of serum glutamine, serine, tryptophan, pyrimidine, and pyruvate were the most relevant metabolic pathways involved in reducing oxidative stress levels by Erchen decoction during DPDRS treatment; especially, mitochondrial redox homeostasis maintenance in endothelial cells may be crucial. In this work, the therapeutic potential of Erchen decoction for reducing the oxidative stress level in DPDRS was demonstrated; however, its in-depth mechanism is worth further exploration.
Topics: Mice; Animals; Endothelial Cells; Drugs, Chinese Herbal; Metabolomics; Chromatography, Liquid; Mass Spectrometry; Cholesterol, LDL; Dyslipidemias; Oxidative Stress
PubMed: 37087794
DOI: 10.1016/j.phymed.2023.154808