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The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis.
No full textNatural proteins often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. Here, we show that the efficient folding and assembly of the five-domain homotetramer β-galactosidase is obligatorily coupled to its synthesis on the ribosome, and we define the underlying mechanisms. During refolding from a denaturant, maturation of the catalytic domain is frustrated. Assembly outpaces monomer folding, and non-native oligomers accumulate. Efficient de novo folding is characterized by segmental domain folding, shaped by the binding of a nascent amphipathic helix to a cryptic pocket on uL23 on the ribosome surface. Homomer assembly also initiates cotranslationally via recruitment of a full-length subunit to the nascent polypeptide, and the failure to do so results in misassembly. Our results reveal how the ribosome can dictate the timing of folding and assembly to enable efficient biogenesis of a topologically complex protein
JAK-STAT and IL-17 pathway dysregulation underlies persistent immune dysfunction in ART-experienced people living with HIV in Ghana
No full textIntroduction
Chronic immune activation and inflammation are central to HIV pathogenesis and persist despite antiretroviral therapy (ART), contributing to non-AIDS comorbidities. The HIV epidemic in West Africa is distinct, marked by the coexistence of HIV-1, HIV-2 in circulation as well as recombinant forms, yet immune responses in this region remain under-investigated. This study examined how ART modulates cytokine and chemokine signaling in Ghanaian people living with HIV (PLWH), with emphasis on biomarkers of immune dysfunction and treatment response.
Methods
Plasma concentrations of 25 cytokines and chemokines were quantified using Luminex multiplex assays in 247 participants: ART-naïve (n=141), post-ART at 6-months (n=52) and 12-months (n=23), ART-experienced (n=74), and HIV-negative controls (n=32). Differentially expressed cytokines, cytokine network analysis, and pathway enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed using R-anchored packages. Correlations between cytokine levels and viral load were also evaluated. Cox proportional hazards regression was applied to identify biomarker of HIV disease progression and predictive modelling using Least Absolute Shrinkage and Selection Operator (LASSO) regression, Random Forest (RF), and Gradient Boosting Machine (GBM).
Results
ART-naïve individuals exhibited elevated pro-inflammatory (IL-6, IL-12/IL-23p40, IL-2, IL-15, IL-2R), and chemotactic (MCP-1, IP-10, MIG) cytokines, alongside reduced IL-1β and IL-1Ra. ART significantly reduced inflammatory cytokines, but paradoxically increased RANTES and Eotaxin. IL-1Ra emerged as the central node in cytokine interaction networks, while IP-10 positively and RANTES negatively correlated with viral load. Lower IL-1β and IL-10 levels predicted virologic control, whereas elevated GM-CSF was linked to persistent viraemia. Machine learning modelling identified RANTES, IP-10, IL-12/IL-23p40, IL-7, and IL-2R as the strongest predictors of viral load. Pathway enrichment analysis revealed upregulation of chemokine-mediated signaling and eosinophil chemotaxis, but downregulation of leukocyte activation, IL-17, and JAK-STAT signaling.
Conclusion
ART attenuates systemic inflammation and partially restores immune balance in PLWH in Ghana, but recovery remains functionally dysregulated, with persistent chemotactic signaling and impaired mucosal and JAK-STAT–mediated immunity. IL-1β, IL-10, GM-CSF, RANTES, and IP-10 emerge as prognostic markers of disease progression and potential targets for adjunctive immunotherapies. These findings underscore the need for immune-modulatory strategies to optimize ART outcomes in West Africa
Plasmodium ARK1 regulates spindle formation during atypical mitosis and forms a divergent chromosomal passenger complex.
No full textMitosis in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis shows that ARK1 forms the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two highly divergent inner centromere proteins (INCENPs), which we term INCENP-A and INCENP-B, and lacking the canonical chromatin-targeting subunits Survivin and Borealin. Comparative genomics suggests that apicomplexan INCENPs arose through recurrent lineage-specific duplications, reflecting an evolutionary rewiring of CPC architecture in this eukaryotic lineage. Together, these findings reveal key adaptations in Plasmodium mitosis involving ARK1 and its INCENP scaffolds, and identify the ARK1-INCENP interface as a potential multistage target for antimalarial intervention
Datasets associated with: Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry
No full textDatasets associated with: Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry., Calcraft, T., Stanke-Scheffler, N., Nans, A., Lindemann, D., Taylor, I. A., & Rosenthal, P. B. (2024). Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry. Cell, 187(16), 4213-4230.</p
Quantifying rate-limiting genetic variation in breast and ovarian tumourigenesis.
No full textBACKGROUND: The number and type of genetic alterations required to initiate breast and ovarian cancer remain unclear. While germline BRCA1/2 carriers show markedly elevated cancer risk, it is uncertain whether point mutations or copy number alterations constitute the rate-limiting events of tumourigenesis. METHODS: We developed a statistical framework extending prior incidence-mutation models to estimate the minimal number and type of driver events required for cancer initiation. Somatic mutation and copy-number data from >3000 breast and ovarian cancers in TCGA and METABRIC were compared between germline BRCA1/2 carriers and non-carriers matched on subtypes. Results were validated through analyses of evolutionary timing data, as well as single-cell whole genome sequencing (scWGS) data of genetically-engineered and patient-derived cancer/pre-cancerous cells. FINDINGS: Deletions, rather than single-nucleotide variants (SNVs), emerged as the likely rate-limiting events. Modeling indicated that 1-3 deletions are sufficient to initiate tumourigenesis, whereas SNVs alone could not explain observed incidence ratios. BRCA1/2-driven and sporadic tumours converged on similar deletion profiles, including early recurrent deletions of chromosomes 13q and 17, though carriers accumulated them more rapidly. INTERPRETATION: Deletion-associated chromosomal instability likely represents the central trigger for breast and ovarian cancer initiation. These results explain why certain somatic driver mutations detected in normal tissues may not predict malignant progression, and that early detection strategies should instead prioritize testing deletions as potential biomarkers. FUNDING: NIH/NCI (P30CA016042; 1U01CA214194-01), NIH NIGMS (R35GM138113, 2R35GM138113), ACS (RSG-22-115-01-DMC), CIHR Vanier Fellowship, and the Francis Crick Institute with core funding from Cancer Research UK, UK Medical Research Council, and Wellcome Trust
CRISPR-Cas9 screening identifies KRAS-induced COX2 as a driver of immunotherapy resistance in lung cancer.
No full textDatasets associated with CRISPR-Cas9 screening identifies KRAS-induced COX2 as a driver of immunotherapy resistance in lung cancer. Boumelha, J., de Castro, A., Bah, N., Cha, H., de Carné Trécesson, S., Rana, S., ... & Downward, J. (2024). CRISPR–Cas9 screening identifies KRAS-induced COX2 as a driver of immunotherapy resistance in lung cancer. Cancer research, 84(14), 2231-2246.</p
The genetic and biochemical basis of human leading strand synthesis.
No full textThe maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Polε). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Polε we define a genetic map of the factors required to support Polε function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Polε activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 and the CHTF18-RFC2/5 complex. By combining cell biology, structural modelling and biochemistry, we define the existence of two tiers of regulation of Polε processivity: leading strand-specific loading of PCNA by CHTF18-RFC2/5 and "gripping" of newly synthesised dsDNA by POLE3-POLE4. The combined loss of these functions is incompatible with leading strand synthesis and viability. In summary, we describe the biochemical basis of human leading strand synthesis and the consequence of its dysfunction in genome stability
Meta-unstable mRNAs in activated CD8+ T cells are defined by interlinked AU-rich elements and m6A mRNA methylation.
No full textCD8+ T cells can rapidly produce effector molecules following activation. This activation triggers rapid changes in gene expression that rely on the control of mRNA levels via multiple mechanisms, including RNA modifications. N6-methyladenosine (m6A) is an abundant post-transcriptional modification that promotes the decay of messenger RNAs in the cytosol. However, how recognition of m6A sites is integrated with other regulatory mechanisms that alter the fate of immunoregulatory mRNAs in CD8+ T cells remains unexplored. Here, we apply the m6A-iCLIP and GLORI methods to identify the importance of m6A sites flanked by AU-rich elements (AREs) within the 3'UTRs of CD8+ T cell mRNAs. Presence of such ARE-flanking m6A motifs predicts meta-unstable mRNAs that rapidly decay upon CD8+ T cell activation. We demonstrate interdependent effects of mutations in the identified AREs and RRACHs on TNF mRNA stability. The ARE-flanking m6A sites in these mRNAs show particularly high iCLIP crosslinking of YTHDF proteins, which are also identified by proteomic interactome analyses along with additional novel RNA-binding proteins. Our study reveals a crosstalk between m6A and ARE-dependent mechanisms in CD8+ T cells, providing new approaches for modulating mRNA decay in T cell activation
Integrin-dependent neutrophil slowing reduces lung perfusion and supports metastasis in a model of breast cancer.
No full textNeutrophils are critical in establishing a tumor-cell-nurturing and immunosuppressive pulmonary "pre-metastatic" niche in breast cancer. The localization and behavior of these neutrophils is, however, not well described. Using multiplexed imaging to investigate the pre-metastatic lung in a spontaneously metastatic mammary cancer model, we uncover that neutrophils with impaired intravascular motility congest the capillaries of pre-metastatic lungs. Slowed neutrophil transit is reversed by activating β2 integrin with an antibody and can be recapitulated by treating non-tumor-bearing mice with G-CSF. Neutrophil congestion causes a reduction of intravenously injected microbeads in the lung, suggestive of lower perfusion. In a model where tumor cells are injected intravenously into mammary-cancer-bearing Rag1-deficient mice, we observe lower lung experimental metastasis burdens after activating β2 integrins. Overall, our study proposes that integrin-mediated neutrophil congestion of the alveolar capillaries contributes to the pulmonary pre-metastatic niche
The thymus regeneration paradox: The search for stemness in an involuting organ.
No full textThe thymus is emerging as a model for studying organ regeneration and stem cell biology. While research has long focused on how antigen-presenting cells shape the T cell repertoire, recent discoveries unveil a far richer cellular landscape that challenges long-held views of thymus structure and function. This review traces the history of early thymic reconstitution assays, the paradigm of clonal stem cells and serial transplantation, assessing evidence for "stemness" within the thymus. A key focus is the paradox that an involuting thymus retains cells able to expand in culture and reconstitute organ function. We differentiate embryonic/fetal thymus development from postnatal homeostasis, emphasizing how the potency of epithelial progenitor/stem cells shifts with age or upon injury. The role of mesenchymal/interstitial cells and the extracellular milieu is considered alongside advances in organ reconstruction. We outline major unsolved questions in the field: thymus regeneration after childhood; the minimal components required to generate functional naïve T cells outside the body; and the potential of next-generation humanized mouse models to interrogate immune tolerance and novel immunotherapies. We argue that thymus research is entering a new era, one in which understanding and harnessing thymus regenerative potential could yield transformative advances in both basic science and clinical applications