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Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.

The discovery of long non-coding RNAs (lncRNAs) has provided a new perspective on the centrality of RNA in gene regulation and genome organization. Here, we screened for lncRNAs with putative functions in the host response to single-stranded RNA respiratory viruses. We identify CARINH as a conserved cis-acting lncRNA up-regulated in three respiratory diseases to control the expression of its antisense gene IRF1, a key transcriptional regulator of the antiviral response. CARINH and IRF1 are coordinately increased in the circulation of patients infected with human metapneumovirus, influenza A virus, or SARS-CoV-2, and in macrophages in response to viral infection or TLR3 agonist treatment. Targeted depletion of CARINH or its mouse ortholog Carinh in macrophages reduces the expression of IRF1/Irf1 and their associated target gene networks, increasing susceptibility to viral infection. Accordingly, CRISPR-mediated deletion of Carinh in mice reduces antiviral immunity, increasing viral burden upon sublethal challenge with influenza A virus. Together, these findings identify a conserved role of lncRNA CARINH in coordinating interferon-stimulated genes and antiviral immune responses.

Mechanical stresses, including compression, arise during cancer progression. In solid cancer, especially breast and pancreatic cancers, the rapid tumor growth and the environment remodeling explain their high intensity of compressive forces. However, the sensitivity of compressed cells to targeted therapies remains poorly known. In breast and pancreatic cancer cells, pharmacological PI3K inactivation decreased cell number and induced apoptosis. These effects were accentuated when we applied 2D compression forces in mechanically responsive cells. Compression selectively induced the overexpression of PI3K isoforms and PI3K/AKT pathway activation. Furthermore, transcriptional effects of PI3K inhibition and compression converged to control the expression of an autophagy regulator, GABARAP, whose level was inversely associated with PI3K inhibitor sensitivity under compression. Compression alone blocked autophagy flux in all tested cells, whereas inactivation of basal PI3K activity restored autophagy flux only in mechanically non-responsive compressed cells. This study provides direct evidence for the role of the PI3K/AKT pathway in compression-induced mechanotransduction. PI3K inhibition promotes apoptosis or autophagy, explaining PI3K importance to control cancer cell survival under compression.

This study investigated the molecular basis of disease severity in acute pyelonephritis (APN), a common and potentially life-threatening bacterial infection. Two cohorts of infants with febrile urinary tract infection were included. Renal involvement was defined by DMSA scans and molecular disease determinants by gene expression analysis and proteomic screens, at diagnosis and after 6 mo. Innate immune hyper-activation, systemically and locally in the urinary tract, was defined as a cytokine storm. Neutrophil degranulation and renal toxicity genes were strongly regulated, with overexpression in the APN group (first DMSA+). Adaptive immune attenuation in the APN group further supported the notion of an immune imbalance. DNA exome genotyping identified APN and febrile urinary tract infection as genetically distinct and scarring associated genes, but the activation of renal toxicity genes during acute infection was unrelated to the development of renal scarring. The results define APN as a hyper-inflammatory disorder with the characteristics of a cytokine storm combined with adaptive immune attenuation. The findings are consistent with innate immune dysfunctions and neutrophil disorders identified as determinants of APN susceptibility in genetic models.

A G4C2 hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore, decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study, we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2, which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1, but to a greater extent RuvBL2, reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines, primary neurons from the C9-500 transgenic mouse model, and patient-derived iPSC motor neurons. In vivo, we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in our Drosophila model was sufficient to rescue a number of DPR-related motor phenotypes. Thus, modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD.

Low oxygen tension (PO₂), characterizes the tissue environment of tumors. The colorectal tumor line Colo205, grown under reduced oxygen tension expresses a novel αDβ1 integrin, which forms a cell surface complex with hemoglobin δ. This resulted in high local affinity for oxygen, which increased cell adhesion as compared with cells grown under normal oxygen tension. Staining with antibodies to the integrin αD polypeptide and hemoglobin δ, and transfection with cDNAs for GFP-hemoglobin δ and mCherry-αD, showed co-localization of αD and hemoglobin δ. Antibodies to αD and β1 integrins, an RGD peptide, and an αDβ1 binding peptide from hemoglobin δ, blocked the αDβ1-hemoglobin interaction and lowered oxygen consumption. Downregulation of integrin αD or hemoglobin δ expression inhibited cell proliferation in hypoxia. The very frequent expression of complexes between αDβ1 and hemoglobin δ on the cell surface offers potential diagnostic and therapeutic targets in colorectal cancer.

Multisubunit protein complexes are central to many cellular processes, and studying their activities and structures in vitro requires reconstitution via recombinant expression and purification. Obtaining targets at sufficient purity and scale typically involves screening several protein variants and expression hosts. Existing cloning strategies enable co-expression but are often time-consuming, labor-intensive, and host-specific, or involve error-prone steps. We present a novel vector set and assembly strategy to overcome these limitations, enabling expression construct generation for multisubunit complexes in a single step. This modular system can be extended to additional hosts or include new tags. We demonstrate its utility by constructing expression vectors for structural maintenance of chromosomes complexes in various hosts, streamlining workflows, and improving productivity.

The brain-related phenotypes observed in 22q11.2 deletion syndrome (DS) patients are highly variable, and their origin is poorly understood. Changes in brain metabolism might contribute to these phenotypes, as many of the deleted genes are involved in metabolic processes, but this is unknown. This study shows for the first time that Tbx1 haploinsufficiency causes brain metabolic imbalance. We studied two mouse models of 22q11.2DS using mass spectrometry, nuclear magnetic resonance spectroscopy, and transcriptomics. We found that Tbx1 ^+/- mice and Df1/⁺ mice, with a multigenic deletion that includes Tbx1, have elevated brain methylmalonic acid, which is highly brain-toxic. Focusing on Tbx1 mutants, we found that they also have a more general brain metabolomic imbalance that affects key metabolic pathways, such as glutamine-glutamate and fatty acid metabolism. We provide transcriptomic evidence of a genotype-vitamin B12 treatment interaction. In addition, vitamin B12 treatment rescued a behavioural anomaly in Tbx1 ^+/- mice. Further studies will be required to establish whether the specific metabolites affected by Tbx1 haploinsufficiency are potential biomarkers of brain disease status in 22q11.2DS patients.

The introduction of antiretroviral therapy has markedly improved the management of HIV-associated neurocognitive disorders (HAND). However, HAND still affects nearly half of HIV-infected individuals, presenting significant challenges to their well-being. This highlights the critical need for a deeper understanding of HAND mechanisms. Among HIV viral proteins, Nef is notable for its multifaceted role in HIV pathogenesis, though its specific involvement in HAND remains unclear. To investigate this, we used a murine model infected with Nef-expressing (EcoHIV) and Nef-deficient (EcoHIVΔNef) murine HIV. Comparative analyses revealed increased neuroinflammation and reduced myelin and neuronal integrity in EcoHIV-infected brains compared with those with EcoHIVΔNef. Both viruses induced astrogliosis, with stronger GFAP activation in Nef-deficient infections. These findings suggest that Nef contributes to neuroinflammation, primarily through microglial targeting and demyelination, although other factors may regulate astrogliosis. Our results indicate that Nef may significantly contribute to neuronal injury in EcoHIV-infected mice, offering insights into Nef-induced neuropathology in HAND and guiding future research.

Actin and microtubules are essential cytoskeletal components and coordinate their dynamics through multiple coupling and decoupling mechanisms. However, how actin and microtubule dynamics are decoupled remains incompletely understood. Here, we identified TBC1D3C as a new regulator that can decouple actin filament assembly from microtubule disassembly. We showed that TBC1D3C induces the release of GEF-H1 from microtubules into the cytosol without perturbing microtubule arrays, leading to RhoA activation and actin filament assembly. Mechanistically, we found that TBC1D3C directly binds to GEF-H1, disrupting its interaction with the Tctex-DIC-14-3-3 complex and thereby displacing GEF-H1 from microtubules independently of microtubule disassembly. Super-resolution microscopy and live-cell imaging further confirmed that TBC1D3C triggers GEF-H1 release and actin filament assembly while maintaining microtubule integrity. Therefore, our findings demonstrated that TBC1D3C functions as a direct GEF activator and a novel regulator in decoupling actin assembly from microtubule disassembly, providing new insights into cytoskeletal regulation.