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The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy 1 . Its hyperactivation contributes to disease in numerous organs, including the heart 1 , 2 , although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3β) or stimulates (AKT, ERK and RSK-1) mTORC1 activity 3 – 9 . Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease 10 – 13 . Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2 S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity. Phosphorylation of one of two adjacent serine residues in TSC2 is both required and sufficient for PKG1-mediated cardiac protection against pressure overload in mice; these serine residues provide a genetic tool for the bidirectional regulation of stress-stimulated mTORC1 activity.

Understanding mechanisms of cancer cell death and the resulting effect on disease progression is crucial in cancer biology and the insight will likely offer better options for therapeutic treatment. Nuclear expulsion occurs in apoptotic cancer cells in a peptidylarginine deiminase 4 (Padi4) dependent manner. The resulting tumor cell nuclear expulsion product (TuNEP) promotes the outgrowth of neighboring cancer cells through chromatin-bound protein complexes. It is not clear what the protein compositions and functionalities are in these TuNEPs. In this study, we performed extensive proteomic profiling and identified key TuNEP protein components from mouse and human breast cancer cells as well as human lung cancer cells (4T1, MDA-MB-231, and PC9). We further compared TuNEP- specific proteins with those from apoptotic bodies or NETs from neutrophils. We found an enrichment of cellular adhesion molecules as well as increased citrullination of proteins associated with RNA binding. We showed that cellular adhesion molecules integrin and basigin (BSG) promote the growth of tumor spheroids. Our work revealed the unique TuNEP protein components distinct from neutrophil-derived NETs and shed light on potential mechanisms by which these cancer cell-derived TuNEPs promote tumor progression.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP. Carboxyl terminus of Hsc70-interacting protein (CHIP) is proteostasis regulator. Here the authors show that CHIP-mediated protein turnover is enhanced by PKG-mediated phosphorylation, which results in attenuated cardiac ischemic proteotoxicity.

The inhibition, in mice, of the phosphodiesterase PDE9A, which specifically regulates natriuretic-peptide-coupled cGMP signalling, is independent of nitric oxide and is upregulated in failing human hearts, and can reverse pre-established stress-induced heart disease. A new treatment for heart disease Work in animals has shown that inhibition of phosphodiesterase type 5A (PDE5A) with, for example, Viagra, can protect the heart from pathological stress by preventing the degradation of nitric-oxide-generated cGMP. However, nitric oxide signalling is often depressed in cardiovascular disease, potentially explaining the disappointing results of PDE5A inhibition in clinical trials. David Kass and colleagues now identify an alternative target, PDE9A, which specifically regulates natriuretic-peptide-coupled cGMP signalling, and is independent of nitric oxide. They show that PDE9A is upregulated in failing human hearts and that its inhibition in mice can reverse pre-established stress-induced heart disease. PDE9A inhibitors seem to be well tolerated in humans and are being investigated in clinical trials for neurocognitive disease. The new results suggest that these inhibitors may also find an application in heart disease. Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease 1 , 2 . However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation 3 . Furthermore, although PDE5A regulates nitric-oxide-generated cGMP 4 , 5 , nitric oxide signalling is often depressed by heart disease 6 . PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7 , 8 ) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys 477 and Cys 484 , and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring. Nitric oxide has been shown to target mitochondrial aconitase 2 and pyruvate dehydrogenase to reprogramme macrophage metabolism. Here, the authors extend these findings to show that lipoate is used to generate nitroxyl in this process.

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8 + T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block. These mutant CD8 + T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rβ and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8 + T cells. The importance of STAT5 tyrosine phosphorylation on T cells has not been investigated in vivo. Here the authors generate STAT5A and STAT5B tyrosine mutant knockin mice and show that Stat5a/b tyrosine is required for maximal IL-2 signaling and CD8 + T cell proliferation.

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling the selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) such as USP5 and USP14. Combined with a ubiquitin-based DUB ABP, the USP18 ABP is employed in a chemoproteomics screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes. USP18 is a multifunctional cysteine protease involved in IFN pathways. Here, the authors report chemical probes and chemoproteomics platforms that allow to detect and perturb USP18 activity in complex biological systems with the potential to unravel important (de)ISGylation-dependent processes.

Duchenne muscular dystrophy (DMD) is an X-linked disorder with dystrophin loss that results in skeletal and cardiac muscle weakening and early death. Loss of the dystrophin–sarcoglycan complex delocalizes nitric oxide synthase (NOS) to alter its signaling, and augments mechanosensitive intracellular Ca2+ influx. The latter has been coupled to hyperactivation of the nonselective cation channel, transient receptor potential canonical channel 6 (Trpc6), in isolated myocytes. As Ca2+ also activates NOS, we hypothesized that Trpc6 would help to mediate nitric oxide (NO) dysregulation and that this would be manifest in increased myocardial S-nitrosylation, a post-translational modification increasingly implicated in neurodegenerative, inflammatory, and muscle disease. Using a recently developed dual-labeling proteomic strategy, we identified 1,276 S-nitrosylated cysteine residues [S-nitrosothiol (SNO)] on 491 proteins in resting hearts from a mouse model of DMD (dmdmdx:utrn+/−). These largely consisted of mitochondrial proteins, metabolic regulators, and sarcomeric proteins, with 80%of them also modified in wild type (WT). S-nitrosylation levels, however, were increased in DMD. Genetic deletion of Trpc6 in this model (dmdmdx:utrn+/−:trpc6−/−) reversed ∼70% of these changes. Trpc6 deletion also ameliorated left ventricular dilation, improved cardiac function, and tended to reduce fibrosis. Furthermore, under catecholamine stimulation, which also increases NO synthesis and intracellular Ca2+ along with cardiac workload, the hypernitrosylated state remained as it did at baseline. However, the impact of Trpc6 deletion on the SNO proteome became less marked. These findings reveal a role for Trpc6-mediated hypernitrosylation in dmdmdx:utrn+/− mice and support accumulating evidence that implicates nitrosative stress in cardiac and muscle disease.

Most tumor cells undergo apoptosis in circulation and at the metastatic organ sites due to host immune surveillance and a hostile microenvironment. It remains to be elucidated whether dying tumor cells have a direct effect on live tumor cells during the metastatic process and what the underlying mechanisms are. Here we report that apoptotic cancer cells enhance the metastatic outgrowth of surviving cells through Padi4-mediated nuclear expulsion. Tumor cell nuclear expulsion results in an extracellular DNA–protein complex that is enriched with receptor for advanced glycation endproducts (RAGE) ligands. The chromatin-bound RAGE ligand S100a4 activates RAGE receptors in neighboring surviving tumor cells, leading to Erk activation. In addition, we identified nuclear expulsion products in human patients with breast, bladder and lung cancer and a nuclear expulsion signature correlated with poor prognosis. Collectively, our study demonstrates how apoptotic cell death can enhance the metastatic outgrowth of neighboring live tumor cells.

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, in this paper, the authors examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). They found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β-dependent phosphorylation at many of the same sites observed with CRT. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.