The N-terminal region of the ubiquitin regulatory x (UBX) domain-containing Protein 1 (UBXD1) modulates interdomain communication within the valosin-containing Protein p97

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors

Regulation of lysosome integrity and lysophagy by the ubiquitin-conjugating enzyme UBE2QL1

Lysosomal membrane permeabilization or full rupture of lysosomes is a common and severe stress condition that is relevant for degenerative disease, infection and cancer. Cells respond with extensive ubiquitination of damaged lysosomes, which triggers selective macroautophagy/autophagy of the whole organelle, termed lysophagy. We screened an siRNA library targeting human E2-conjugating enzymes and identified UBE2QL1 as critical for efficient lysosome ubiquitination after chemically-induced lysosomal damage. UBE2QL1 translocates to lysosomes upon damage and associates with autophagy regulators. Loss of UBE2QL1-mediated ubiquitination reduces association of the autophagy receptor SQSTM1/p62 and the LC3-decorated phagophore, and prevents recruitment of the ubiquitin-targeted AAA-ATPase VCP/p97 that facilitates lysophagy. Even in unchallenged cells, UBE2QL1 depletion leads to MTOR dissociation and TFEB activation, and mutation of the homolog UBC-25 destabilizes lysosomes in C. elegans, indicating that UBE2QL1 is critical for maintaining lysosome integrity in addition to lysophagy

The de-ubiquitylating enzyme DUBA is essential for spermatogenesis in Drosophila

De-ubiquitylating enzymes (DUBs) reverse protein ubiquitylation and thereby control essential cellular functions. Screening for a DUB that counteracts caspase ubiquitylation to regulate cell survival, we identified the Drosophila ovarian tumour-type DUB DUBA (CG6091). DUBA physically interacts with the initiator caspase death regulator Nedd2-like caspase (Dronc) and de-ubiquitylates it, thereby contributing to efficient inhibitor of apoptosis-antagonist-induced apoptosis in the fly eye. Searching also for non-apoptotic functions of DUBA, we found that Duba-null mutants are male sterile and display defects in spermatid individualisation, a process that depends on non-apoptotic caspase activity. Spermatids of DUBA-deficient flies showed reduced caspase activity and lack critical structures of the individualisation process. Biochemical characterisation revealed an obligate activation step of DUBA by phosphorylation. With genetic rescue experiments we demonstrate that DUBA phosphorylation and catalytic activity are crucial in vivo for DUBA function in spermatogenesis. Our results demonstrate for the first time the importance of de-ubiquitylation for fly spermatogenesis

Macroscopic Displacement Reaction of Copper Sulfide in Lithium Solid-State Batteries

Copper sulfide (CuS) is an attractive electrode material for batteries, thanks to its intrinsic mixed conductivity, ductility and high theoretical specific capacity of 560 mAh g−1. Here, CuS is studied as cathode material in lithium solid-state batteries with an areal loading of 8.9 mg cm−2 that theoretically corresponds to 4.9 mAh cm−2. The configuration of the cell is LiLi3PS4[CuS (70 wt%) + Li3PS4 (30 wt%)]. No conductive additive is used. CuS undergoes a displacement reaction with lithium, leading to macroscopic phase separation between the discharge products Cu and Li2S. In particular, Cu forms a network of micrometer-sized, well-crystallized particles that seems to percolate through the electrode. The formed copper is visible to the naked eye. The initial specific discharge capacity at 0.1 C is 498 mAh g(CuS)−1, i.e., 84% of its theoretical value. The initial Coulomb efficiency (ICE) reaches 95%, which is higher compared to standard carbonate-based liquid electrolytes for the same cell chemistry (≈70%). After 100 cycles, the specific capacity reaches 310 mAh g(CuS)−1. With the current composition, the cell provides 58.2 Wh kg−1 at a power density of 7 W kg−1, which is superior compared to other transition metal sulfide cathodes.Peer Reviewe

Lessons from the frontline: Documenting the pandemic emergency care experience from the Pacific region - Infrastructure and equipment

BACKGROUND: The COVID-19 pandemic highlighted challenges for all health systems worldwide. This research aimed to explore the impact of COVID-19 across the Pacific especially with regards to emergency care (EC) and clinicians' preparations and responses. METHODS: A collaboration of Australia and Pacific researchers conducted prospective qualitative research over 18 months of the pandemic. In this three phase study data were gathered from Emergency Clinicians and stakeholders through online support forums, in-depth interviews and focus groups. A phenomenological methodological approach was employed to explore the lived experience of participants. This paper discusses the findings of the study regarding the EC building block of 'Infrastructure and Equipment.' FINDINGS: Pre-existing infrastructure and equipment were not sufficient to help control the pandemic. Adequate space and correct equipment were essential needs for Pacific Island emergency clinicians, with donations, procurement and local ingenuity required for suitable, sustainable supplies and facilities. Adequate personal protective equipment (PPE) conferred a sense of security and increased Health Care Workers willingness to attend to patients. INTERPRETATION: Investing in adequate infrastructure and appropriate equipment is crucial for an effective response to the COVID-19 pandemic. The sustainability of such investments in the Pacific context is paramount for ongoing EC and preparation for future surge responses and disasters. FUNDING: Phases 1 and 2A of this study were part of an Epidemic Ethics/World Health Organization (WHO) initiative, supported by Foreign, Commonwealth and Development Office/Wellcome Grant 214711/Z/18/Z. Co-funding for this research was received from the Australasian College for Emergency Medicine Foundation via an International Development Fund Grant

Lessons from the frontline: Documenting the experiences of Pacific emergency care clinicians responding to the COVID-19 pandemic

UNLABELLED: Low- and middle-income countries (LMICs) across the Pacific region have been severely impacted by the COVID-19 pandemic, and emergency care (EC) clinicians have been on the frontline of response efforts. Their responsibilities have extended from triage and clinical management of patients with COVID-19 to health system leadership and coordination. This has exposed EC clinicians to a range of ethical and operational challenges.This paper describes the context and methodology of a rapid, collaborative, qualitative research project that explored the experiences of EC clinicians in Pacific LMICs during the COVID-19 pandemic. The study was conducted in three phases, with data obtained from online regional EC support forums, key informant interviews and focus group discussions. A phenomenological approach was adopted, incorporating a hybrid inductive and deductive thematic analysis. Research findings, reported in other manuscripts in this collection, will inform multi-sectoral efforts to improve health system preparedness for future public health emergencies. FUNDING: Epidemic Ethics/World Health Organization (WHO) initiative, supported by Foreign, Commonwealth and Development Office/Wellcome Grant 214711/Z/18/Z (Phases 1 and 2A) and an Australasian College for Emergency Medicine Foundation International Development Fund Grant

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gs alpha. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gs alpha induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gs alpha -dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gs alpha -PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.</p