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Background A subpopulation of endothelial progenitor cells called endothelial colony‐forming cells (ECFCs) may offer a platform for cellular assessment in clinical studies because of their remarkable angiogenic and expansion potentials in vitro. Despite endothelial cell function being influenced by cardiovascular risk factors, no studies have yet provided a comprehensive proteomic profile to distinguish functional (ie, more angiogenic and expansive cells) versus dysfunctional circulating ECFCs of young adults. The aim of this study was to provide a detailed proteomic comparison between functional and dysfunctional ECFCs. Methods and Results Peripheral blood ECFCs were isolated from 11 subjects (45% men, aged 27±5 years) using Ficoll density gradient centrifugation. ECFCs expressed endothelial and progenitor surface markers and displayed cobblestone‐patterned morphology with clonal and angiogenic capacities in vitro. ECFCs were deemed dysfunctional if <1 closed tube formed during the in vitro tube formation assay and proliferation rate was <20%. Hierarchical functional clustering revealed distinct ECFC proteomic signatures between functional and dysfunctional ECFCs with changes in cellular mechanisms involved in exocytosis, vesicle transport, extracellular matrix organization, cell metabolism, and apoptosis. Targeted antiangiogenic proteins in dysfunctional ECFCs included SPARC (secreted protein acidic and rich in cysteine), CD36 (cluster of differentiation 36), LUM (lumican), and PTX3 (pentraxin‐related protein PYX3). Conclusions Circulating ECFCs with impaired angiogenesis and expansion capacities have a distinct proteomic profile and significant phenotype changes compared with highly angiogenic endothelial cells. Impaired angiogenesis in dysfunctional ECFCs may underlie the link between endothelial dysfunction and cardiovascular disease risks in young adults.
AbstractThe histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define a novel therapeutic strategy for targeting the loss of SETD2 in cancer.Significance StatementThe histone-modifying enzyme SETD2 has emerged as an important tumour suppressor in a number of different cancer types, identifying it as a promising therapeutic target. The concept of synthetic lethality, a genetic interaction in which the simultaneous loss of two genes or pathways that regulate a common essential process renders the cell nonviable, is a valuable tool for killing cancer cells that have known mutations. In this study, we conducted a synthetic lethality screen for compounds that specifically target SETD2-deficient cancer cells. The top hit, a compound called RITA, reduces cell viability and induces cell death only in the context of SETD2 loss, thereby highlighting a potential novel therapeutic strategy for treating SETD2-deficient cancers.
HLA-E–restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities
Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)–restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1–specific, HLA-E–restricted T cells have not been observed in HIV-1–infected individuals. Here, HLA-E–restricted, HIV-1–specific CD8+ T cells were primed in vitro. These T cell clones and allogeneic CD8+ T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4+ T cells in vitro. Vaccine induction of efficacious HLA-E–restricted HIV-1–specific T cells should therefore be possible.
Incoming HIV virion-derived Gag Spacer Peptide 2 (p1) is a target of effective CD8+ T cell antiviral responses.
Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial" HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A∗02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8+ T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone progressive change, with variants conferring advantage rapidly becoming dominant lineages, e.g., B.1.617. With apparent increased transmissibility, variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the United Kingdom. Here we study the ability of monoclonal antibodies and convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2, complement this with structural analyses of Fab/receptor binding domain (RBD) complexes, and map the antigenic space of current variants. Neutralization of both viruses is reduced compared with ancestral Wuhan-related strains, but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2, suggesting that individuals infected previously by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insights for immunization policy with future variant vaccines in non-immune populations.
Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain.
Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.
Isoform resolved measurements of absolute translational efficiency define interplay of HIF and mTOR dysregulation in kidney cancer
AbstractHypoxia inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways orchestrate responses to oxygen and nutrient availability. These pathways are frequently dysregulated in cancer, but their interplay is poorly understood, in part because of difficulties in simultaneous measurement of global and mRNA-specific translation. Here we describe a workflow for measurement of absolute translational efficiency of mRNAs resolved by their transcription start sites (TSSs). Its application to kidney cancer cells revealed a remarkable extent of translational reprogramming by mTOR, strongly affecting many metabolic enzymes and pathways. By contrast, global effects of HIF on translation were limited, and we did not observe reported translational activation by HIF2A. In contrast, HIF-dependent alterations in TSS usage were associated with robust changes in translational efficiency in a subset of genes. Analyses of the interplay of HIF and mTOR revealed that specific classes of HIF1A and HIF2A transcriptional target gene manifest markedly different sensitivity to mTOR, in a manner that supports combined use of HIF2A and mTOR inhibitors in treatment of kidney cancer.
The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.
Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences
SummaryDespite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the Alpha variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.Graphical AbstractIn briefBy detecting nearly all individual SARS-CoV-2 RNA molecules we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain.HighlightsSingle molecule quantification of SARS-CoV-2 replication uncovers early infection kineticsThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replicationGenomic RNA is stable and persistent during the initial stages of infectionAlpha (B.1.1.7) variant of concern replicates more slowly than the Victoria strain
PD-1 Expression Status on CD8+ Tumour Infiltrating Lymphocytes Associates With Survival in Cervical Cancer.
Despite the expansion of PD-1 checkpoint blockade to multiple types of cancer, whether the programmed cell death 1 (PD-1) expression status on CD8+ tumour infiltrating lymphocytes (TILs) could be a prognostic factor in cervical cancer is still unclear. In this study, we performed ex vivo phenotypic analysis of PD-1 expression on CD8+ TILs by flow cytometry from 47 treatment-naïve cervical cancer patients. With a median follow-up of 26.1 months (95% confidence interval [CI], 24-28.2 months), we then linked the quantitative cellular expression results to progression-free survival and overall survival. Based on the intensity of PD-1 expression, we further categorised the cervical cancer patients into PD-1high expressers (29.8%, 14/47) and PD-1low expressers (70.2%, 33/47). Multivariate analysis revealed that PD-1high expressers are correlated with early recurrence (HR, 5.91; 95% CI, 1.03-33.82; P= 0.046). Univariate analysis also demonstrated that PD-1high expressers are associated with poor overall survival in cervical cancer (HR, 5.365; 95% CI, 1.55-18.6; P=0.008). Moreover, our study also demonstrated that CD8+/CD4+ TIL ratio and HPV infection status are risk factors for early relapse and mortality in cervical cancer patients. In conclusion, this study confirms that PD-1 expression status is an independent prognostic factor for progression free survival in cervical cancer. These findings could be important in predicting the relapse of cervical cancer as a cellular diagnosis method and could be important knowledge for the selection of prospective PD-1 blockade candidates.
Deceased donor kidney degradomics suggest cytoskeletal proteolytic processes impact post-transplant graft function
ABSTRACTBackgroundIn brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys that adversely impacts the quality of grafts. Here, we hypothesized that DBD kidneys may undergo proteolytic processes that renders grafts susceptible to post-transplant dysfunction.Material & MethodsUsing mass spectrometry and immunoblotting analyses, we profiled degradation patterns of cytoskeletal proteins in deceased (n=55) and living (n=10) donor kidneys.ResultsWe found that in DBD kidneys, key podocyte cytoskeletal proteins had been proteolytically cleaved. Generated degradation profiles were not associated to donor related demographic and clinical factors but were associated to suboptimal post-transplant function. Strikingly, α-actinin -4 and Talin-1 degradation profiles were not observed in circulatory-death or living-donor kidneys. As Talin-1 is a specific proteolytic target of Calpain-1, we investigated a potential trigger of Calpain activation and Talin-1 degradation using ex-vivo precision-cut human kidney slices and in-vitro immortalised human podocytes. Notably, we found that Transforming-Growth Factor-β (TGF-β) activated Calpain-1 and proteolytically cleaved Talin-1 to generate distinct peptide fragments. These peptide fragments were of similar size and matched the degradation patterns observed in DBD kidneys. Talin-1 degradation was prevented in vitro by Calpain-1 inhibition.ConclusionsHere, we provide initial evidence that DBD kidneys are susceptible to cytoskeletal protein degradation that impacts posttransplant kidney function. Subsequent studies should aim to further investigate the link between brain death and activation of proteolytic pathways exploring new therapeutic opportunities.
Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination.
Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons
Abstractα-Synuclein is critical in the pathogenesis of Parkinson’s disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson’s disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson’s disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.
Kawasaki Disease Patient Stratification and Pathway Analysis Based on Host Transcriptomic and Proteomic Profiles.
The aetiology of Kawasaki disease (KD), an acute inflammatory disorder of childhood, remains unknown despite various triggers of KD having been proposed. Host 'omic profiles offer insights into the host response to infection and inflammation, with the interrogation of multiple 'omic levels in parallel providing a more comprehensive picture. We used differential abundance analysis, pathway analysis, clustering, and classification techniques to explore whether the host response in KD is more similar to the response to bacterial or viral infections at the transcriptomic and proteomic levels through comparison of 'omic profiles from children with KD to those with bacterial and viral infections. Pathways activated in patients with KD included those involved in anti-viral and anti-bacterial responses. Unsupervised clustering showed that the majority of KD patients clustered with bacterial patients on both 'omic levels, whilst application of diagnostic signatures specific for bacterial and viral infections revealed that many transcriptomic KD samples had low probabilities of having bacterial or viral infections, suggesting that KD may be triggered by a different process not typical of either common bacterial or viral infections. Clustering based on the transcriptomic and proteomic responses during KD revealed three clusters of KD patients on both 'omic levels, suggesting heterogeneity within the inflammatory response during KD. The observed heterogeneity may reflect differences in the host response to a common trigger, or variation dependent on different triggers of the condition.