The Multifunctional Sorting Protein PACS-2 Regulates SIRT1-Mediated Deacetylation of p53 to Modulate p21-Dependent Cell-Cycle Arrest

SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here, we demonstrate that the sortingprotein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell-cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2-/- mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell-cycle arrest in PACS-2 knockdown cells. Traffickingstudies revealed that cytoplasmic PACS-2 shuttled to the nucleus, where it interacted with SIRT1 andrepressed SIRT1-mediated p53 deacetylation. Correspondingly, invitro assays demonstrated that PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an invivo mediator of the SIRT1-p53-p21 axis that modulates the DNA damage response

Hysteretic Behavior of Proprotein Convertase 1/3 (PC1/3)

The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis. This is an unprecedented observation in peptidases, but is frequent in regulatory enzymes with physiological relevance. The lag phase of mPC1/3 is dependent on substrate, calcium concentration and pH. This hysteretic behavior may have implications in the physiological processes in which PC1/3 participates and could be considered an additional control step in the peptide hormone maturation processes as for instance in the transformation of proinsulin to insulin

Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling