Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates β1-Ser108 on the regulatory β1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.

It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

Poor access to human left ventricular myocardium is a significant limitation in the study of heart failure (HF). Here, we utilise a carefully procured large human heart biobank of cryopreserved left ventricular myocardium to obtain direct molecular insights into ischaemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), the most common causes of HF worldwide. We perform unbiased, deep proteomic and metabolomic analyses of 51 left ventricular (LV) samples from 44 cryopreserved human ICM and DCM hearts, compared to age-, gender-, and BMI-matched, histopathologically normal, donor controls. We report a dramatic reduction in serum amyloid A1 protein in ICM hearts, perturbed thyroid hormone signalling pathways and significant reductions in oxidoreductase co-factor riboflavin-5-monophosphate and glycolytic intermediate fructose-6-phosphate in both; unveil gender-specific changes in HF, including nitric oxide-related arginine metabolism, mitochondrial substrates, and X chromosome-linked protein and metabolite changes; and provide an interactive online application as a publicly-available resource.

Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogate hepatocyte protein secretion in two models of murine NASH to understand how liver-derived factors modulate lipid metabolism and insulin sensitivity in peripheral tissues. We reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism, and identify arylsulfatase A (ARSA) as a hepatokine that is upregulated in NASH and type 2 diabetes. Mechanistically, hepatic ARSA reduces sulfatide content and increases lysophosphatidylcholine (LPC) accumulation within lipid rafts and suppresses LPC secretion from the liver, thereby lowering circulating LPC and lysophosphatidic acid (LPA) levels. Reduced LPA is linked to improvements in skeletal muscle insulin sensitivity and systemic glycemic control. Hepatic silencing of Arsa or inactivation of ARSA's enzymatic activity reverses these effects. Together, this study provides a unique resource describing global changes in hepatokine secretion in NASH, and identifies ARSA as a regulator of liver to muscle communication and as a potential therapeutic target for type 2 diabetes.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.