Chebychev Trajectory Optimization Program /CHEBYTOP/ Final report

Digital computer program for interplanetary trajectory optimization and variable thrust dat

Phosphorylation Provides a Negative Mode of Regulation for the Yeast Rab GTPase Sec4p

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p

Brd4 activates P-TEFb for RNA polymerase II CTD phosphorylation

The bromodomain protein Brd4 regulates the transcription of signal-inducible genes. This is achieved by recruiting the positive transcription elongation factor P-TEFb to promoters by its P-TEFb interaction domain (PID). Here we show that Brd4 stimulates the kinase activity of P-TEFb for phosphorylation of the C-terminal domain (CTD) of RNA polymerase II over basal levels. The CTD phosphorylation saturation levels, the preferences for pre-phosphorylated substrates, and the phosphorylation specificity for Ser5 of the CTD however remain unchanged. Inhibition of P-TEFb by Hexim1 is relieved by Brd4, although no mutual displacement with the Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence motif similarity to the trans-activating Tat protein from HIV-1, which includes a core RxL motif, a polybasic cluster known as arginine-rich motif, and a C-terminal leucine motif. Mutation of these motifs to alanine significantly diminished the stimulatory effect of Brd4 and fully abrogated its activation potential in presence of Hexim1. Yet the protein was not found to bind Cyclin T1 as Tat, but only P-TEFb with a dissociation constant of 0.5 muM. Our data suggest a model where Brd4 acts on the kinase subunit of P-TEFb to relieve inhibition and stimulate substrate recognition

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVD-directed targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners

Locking GTPases covalently in their functional states

GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya