Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts’ increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition towards ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here we examined the ability of the 8 most prevalent HLA-B27 allotypes (HLA-B*27:02 - HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers in comparison to all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position-59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine-59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine-67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features such as peptide binding and antigen presentation as pivotal mechanisms for disease pathogenesis.

We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.

The PEAK family of pseudokinases, comprising PEAK1-3, play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screen for PEAK1 effectors and identify calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promotes CAMK2 activation in TNBC cells via PLCγ1/Ca²⁺ signalling and direct binding to CAMK2. In turn, CAMK2 phosphorylates PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurpose RA306, a second generation CAMK2 inhibitor. RA306 inhibits PEAK1-enhanced migration and invasion of TNBC cells in vitro and significantly attenuates TNBC xenograft growth and metastasis in a manner mirrored by PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus that integrates Ca²⁺ and tyrosine kinase signals and identify CAMK2 as a therapeutically ‘actionable’ target downstream of PEAK1.

Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.

The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal “membrane trafficking” as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action.