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SUMMARYThe Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy andin silicopredictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings., Exercise training can be therapeutic but how mitochondria respond remains unclear. Here, the authors use multiple omics techniques to reveal a complex network of non-stoichiometric mitochondrial adaptations that are prioritized or deprioritised during different phases of exercise training.

Context Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. Objective We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. Methods We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. Results We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. Conclusion A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.

The four complexes of the mitochondrial respiratory chain are critical for ATP production in most eukaryotic cells. Structural characterisation of these complexes has been critical for understanding the mechanisms underpinning their function. The three proton-pumping complexes, Complexes I, III and IV associate to form stable supercomplexes or respirasomes, the most abundant form containing 80 subunits in mammals. Multiple functions have been proposed for the supercomplexes, including enhancing the diffusion of electron carriers, providing stability for the complexes and protection against reactive oxygen species. Although high-resolution structures for Complexes III and IV were determined by X-ray crystallography in the 1990s, the size of Complex I and the supercomplexes necessitated advances in sample preparation and the development of cryo-electron microscopy techniques. We now enjoy structures for these beautiful complexes isolated from multiple organisms and in multiple states and together they provide important insights into respiratory chain function and the role of the supercomplex. While we as non-structural biologists use these structures for interpreting our own functional data, we need to remind ourselves that they stand on the shoulders of a large body of previous structural studies, many of which are still appropriate for use in understanding our results. In this mini-review, we discuss the history of respiratory chain structural biology studies leading to the structures of the mammalian supercomplexes and beyond.

Cephalopods are known to produce an extensive range of secretions including ink, mucus, and venom. Sepiadariidae, a family of small, benthic bobtail squids, are notable for the high volume of viscous slime they emit when stressed. One species, Sepioloidea lineolata (striped pyjama squid), is covered with glands along the perimeter of the ventral mantle, and these structures are hypothesized to be the source of its slime. Using label-free quantitative proteomics, we analyzed five tissue types (dorsal and ventral mantle muscle, dorsal and ventral epithelium, and ventral mantle glands) and the slime from four individuals. In doing so, we were able to determine the relationship between the slime and the tissues as well as highlight proteins that were specifically identified within the slime and ventral mantle glands. A total of 28 proteins were identified to be highly enriched in slime, and these were composed of peptidases and protease inhibitors. Seven of these proteins contained predicted signal peptides, indicating classical secretion, with four proteins having no identifiable domains or similarity to any known proteins. The ventral mantle glands also appear to be the tissue with the closest overall proteomic composition to the slime; therefore, it is likely that the slime originates, at least in part, from these glands.

Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2; previously called YueF) is a mitochondrial inner membrane/matrix-residing protein and is reported to regulate mitochondrial function. The clinical importance of PYROXD2 has been unclear, and little is known of the protein’s precise biological function. In the present paper, we report biallelic variants in PYROXD2 identified by genome sequencing in a patient with suspected mitochondrial disease. The child presented with acute neurological deterioration, unresponsive episodes, and extreme metabolic acidosis, and received rapid genomic testing. He died shortly after. Magnetic resonance imaging (MRI) brain imaging showed changes resembling Leigh syndrome, one of the more common childhood mitochondrial neurological diseases. Functional studies in patient fibroblasts showed a heightened sensitivity to mitochondrial metabolic stress and increased mitochondrial superoxide levels. Quantitative proteomic analysis demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect. Our findings support the critical role of PYROXD2 in human cells, and suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction, and can plausibly explain the child’s clinical presentation.

SUMMARYMitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings.

Cephalopods are known to produce an extensive range of secretions including ink, mucus, and venom. Sepiadariidae, a family of small, benthic bobtail squids, are notable for the high volume of viscous slime they emit when stressed. One species

Sepioloidea lineolata, the striped pyjama squid (family Sepiadariidae), is a small species of benthic bobtail squid distributed along the Southern Indo-Pacific coast of Australia. Like other sepiadariid squids, it is known to secrete large volumes of viscous slime when stressed. In order to identify key proteins involved in the function of sepiadariid slimes, we compared the slime proteome of Sepioloidea lineolata with that of a closely related species, Sepiadarium austrinum. Of the 550 protein groups identified in Sepioloidea lineolata slime, 321 had orthologs in Sepiadarium austrinum, and the abundance of these (iBAQ) was highly correlated between species. Both slimes were dominated by a small number of abundant proteins, and several of these were short secreted proteins with no homologues outside the class Cephalopoda. No mucins were identified within either species' slime, suggesting that it is structurally distinct from mucin polymer-based gels found in many vertebrate and echinoderm secretions. The extent of N-glycosylation in the slime of Sepioloidea lineolata was also studied via glycan cleavage with Peptide: N-glycosidase F (PNGase-F). Although very few (four) proteins showed strong evidence of N-glycosylation, we found that treatment with PNGase-F led to a slight increase in peptide identification rates compared with controls.

Sepiadarium austrinum, the southern bottletail squid, is a small squid that inhabits soft sediments along Australia's south-east coast. When provoked, it rapidly secretes large volumes of slime, presumably as a form of chemical defense. We analyzed the proteomic composition of this slime using tandem mass spectrometry and transcriptomics and found that it was remarkably complex with 1735 identified protein groups (FDR:0.01). To investigate the chemical defense hypothesis we performed an Artemia toxicity assay and used sequence analysis to search for toxin-like molecules. Although the slime did not appear to be toxic to Artemia we found 13 proteins in slime with the hallmarks of toxins, namely cysteine richness, short length, a signal peptide and/or homology to known toxins. These included three short (80-130AA) cysteine rich secreted proteins with no homology to proteins on the NCBI or UniProt databases. Other protein families found included, CAP, phospholipase-B, ShKT-like peptides, peptidase S10, Kunitz BPTI and DNase II. Quantitative analysis using intensity based absolute quantification (iBAQ via MaxQuant) revealed 20 highly abundant proteins, accounting for 67% of iBAQ signal, and three of these were toxin-like. No mucin homologues were found suggesting that the structure of the slime gel may be formed by an unknown mechanism.This study is the first known instance of a slime secretion from a cephalopod to be analyzed by proteomics methods and is the first investigation of a member of the family Sepiadariidae using proteomic methods. 1735 proteins were identified with 13 of these fitting criteria established for the identification of putative toxins. The slime is dominated by 20 highly abundant proteins with secreted, cysteine rich proteins. The study highlights the importance of 'omics approaches in understanding novel organisms.

Cephalopods are a morphologically diverse molluscan class that includes octopuses, cuttlefishes, squids, and nautiluses. The behavior, morphology, and sometimes aposematic appearance of coleoid cephalopods (octopuses, cuttlefishes, and squids) are highly suggestive of the widespread use of toxins for predation and/or defense. Many cephalopods use a combination of their parrot-like beak and/or toothed radula to inject venomous saliva, thought to be produced in the posterior salivary gland, into prey through a bite wound or a hole drilled into the shell. However, relatively few toxins have been studied to date from only a narrow range of cephalopods. Active components that have been identified from cephalopod posterior salivary gland extracts (or saliva) include neurotoxins such as tetrodotoxin (also found in body tissues), tachykinins and cephalotoxins, biogenic amines such as serotonin and octopamine, and a diverse range of enzymes including serine proteases, phospholipase A2, hyaluronidases, and chitinases. Coleoid cephalopods represent excellent candidates for biodiscovery, being taxonomically distinct from heavily studied venom-producing organisms, and because their venoms appear to be complexmixtures of proteins and smallmolecules. Understanding the evolutionary history of toxicity in cephalopods remains a challenge, with many major taxa remaining unstudied and very little specific functional information available onmost cephalopod toxins. The application of "omics" technologies to research on venoms and other toxic secretions ("venomics") is an important and powerful way of characterizing entire suites of proteinaceous toxins from pure venom or gland extracts in cephalopods and is likely to yield future insights into the evolution of toxicity in this class.