The evolutionarily conserved Ser-Thr kinase mTOR plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phospho-proteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently downregulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

Highlights d Heterogeneity and plasticity of non-parenchymal cells in healthy and NASH liver d Landscape of intrahepatic ligand-receptor signaling at single-cell resolution d Emergence of Trem2+ NASH-associated macrophages (NAMs) in mouse and human NASH d Stellakine secretion and contractile response to vasoactive hormones by HSCs

Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid- and glutamic acid-ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from NAD+ onto substrate proteins. Here, we report a robust NAD+ analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits NELF, a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1, or mutation of the ADP-ribosylation sites on NELF-E, promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family, and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

SUMMARY Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.