THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

Catalytic antibody response to the Staphylococcus aureus virulence factor Efb

Staphylococcus aureus is a globally prevalent pathogen that can cause a wide variety of acute and chronic diseases in both adults and children, in both immune susceptible populations and healthy individuals. Its ability to cause persistent infections has been linked to multiple immune evasion strategies, including Efb-mediated complement inhibition. As new multi-drug-resistant strains emerge, therapeutic alternatives to traditional antibiotics must be developed. These experiments assessed the ability of healthy patient immunoglobulin to cleave Efb and disable the complement-inhibitory properties of Efb in vitro. Levels of immunoglobulin-mediated Efb catalysis varied both between immunoglobulin isoform/isotype and between individuals. Serum IgG showed the strongest catalytic activity of the immunoglobulin isotypes tested. Additionally, IgG hydrolyzed the virulence factor in a way that enabled only minimal binding to the complement component C3b, effectively blocking Efb-mediated inhibition of complement lysis. Salivary IgA and serum IgM did not block Efb-mediated inhibition of complement. Catalytic IgG selectively cleaved Efb and showed no cleavage of a variety of other proteins tested. Catalytic activity of IgG was inhibited by serine protease inhibitors, but not by other protease inhibitors, suggesting a serine-protease mechanism of catalysis. It is proposed that varying concentrations and activity levels of catalytic IgG between healthy individuals and those with current or recurrent S. aureus infections in both adult and pediatric populations be studied in order to assess the potential effectiveness of passive immunization therapy with catalytic monoclonal IgG. ^

Change in permeation rates of acids and bases through limited use protective fabrics after repeated wash cycles

The primary objective of this study was to determine if there is a change in permeation rates when limited use protective fabrics undergo repeated exposure and wash cycles. The null hypothesis of this study was that no substantial change in permeation takes place after the test material is subjected to repeated contact with a strong acid or base and has undergone repeated wash cycles. ^ The materials tested were DuPont Tychem® CPF 3 and CPF 4 fabrics. The challenge chemicals in this study were ninety-eight percent sulfuric acid and fifty percent sodium hydroxide. Permeation testing was conducted utilizing ASTM designation F739-99a Standard Test Method for Resistance of Protective Clothing Materials to Permeation by Liquids or Gases Under Conditions of Continuous Contact. ^ In this study, no change in permeation rates of either challenge chemical was detected for CPF 3 or CPF 4 limited use protective fabrics after repeated exposure and wash cycles. Certain unexposed areas of the fabric suffered structural degradation unrelated to exposure and which may be due to multiple washings.^

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

The Role of K63-linked Ubiquitination Cycles in Akt Kinase Activation

Akt (also known as protein kinase B) serves a central regulator in PI3K/Akt signaling pathways to regulate numerous physiological functions including cell proliferation, survival and metabolism. Akt activation requires the binding of Akt to phospholipid PIP3 on the plasma membrane and subsequent phosphorylation of Akt by its kinases. Growth factor-mediated membrane recruitment of Akt is a crucial step for Akt activation. However, the mechanism of Akt membrane translocation is unclear. Protein ubiquitination is a significant posttranslational modification that controls many biological functions such as protein trafficking and signaling activation. Therefore, we hypothesize that ubiquitination may be involved in Akt signaling activation. We have demonstrated that Akt could be conjugated with non-proteolytic K63-linked ubiquitination by TRAF6 ubiquitin E3 ligase. This modification on Akt was required for membrane recruitment, phosphorylation and activation of Akt in response to growth factor stimulation. The human cancer-associated Akt E17K mutant exhibited an increase in K63-linked ubiquitination, which contributes to the enrichment of membrane recruitment and phosphorylation of Akt. Thus, we conclude that K63-linked ubiquitination is a critical step for oncogenic Akt activation and also involved in human cancer development. Notably, the process of protein ubiquitination can be reversed by deubiquitinating enzymes (DUBs), which play a critical role to terminate signaling activation induced by ubiquitination. To further investigate how ubiquitination cycles regulate Akt activation, we have identified that CYLD as a DUB for Akt, and CYLD inhibited growth factor-induced ubiquitination and activation of Akt. Under serum-depletion condition, CYLD interacts with Akt and keep Akt under inactive state by directly removing K63-linked ubiquitination of Akt. CYLD disassociates with Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. We also demonstrated that CYLD deficiency promoted cancer cell proliferation, survival, glucose metabolism and human prostate cancer development. Therefore, we conclude that CYLD plays a critical role for negatively regulating Akt signaling activation through deubiquitination of Akt. In summary, this study delineated the important mechanism of cycles of ubiquitination and deubiquitination of Akt in regulating membrane translocation and activation of Akt, and TRAF6 and CYLD as central switches for these processes.