Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.     

Muscular dystrophies,the cytoskeleton and cell adhesion

Muscular dystrophies are associated with mutations in genes encoding several classes of proteins. These range from extracellular matrix and integral membrane proteins to cytoskeletal proteins, but also include a heterogeneous group of proteins including proteases, nuclear proteins, and signalling molecules. Muscular dystrophy phenotypes have also become evident in studies on various knockout mice defective in proteins not previously considered or known to be mutated in muscular dystrophies. Some unifying themes are beginning to emerge from all of these data. This review will consider recent advances in our understanding of the molecules involved and bring together data that suggest a role for the cytoskeleton and cell adhesion in muscular dystrophies.     

Segregation analysis of blood pressure and body mass index in a rural US community

To assess evidence for a gene with large effect on systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI), we conducted segregation analyses on 261 nuclear families collected from a rural Caucasian community in Michigan. The families were ascertained through a hypertensive proband. Each phenotype was adjusted for significant covariate effects (e.g., gender and age). We used class D regressive models to conduct the segregation analyses. Our analysis results support the segregation of a major gene for BMI, but not for SBP or DBP. A recessive locus effect provided the best explanation for BMI where approximately 43% of the variance of BMI was due to this gene.     

A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.     

Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.     

Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis

Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1-/- CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.     

Osmoregulation of endothelial nitric-oxide synthase gene expression in inner medullary collecting duct cells. Role in activation of the type A natriuretic peptide receptor

Previously, we showed that increased extracellular tonicity promotes increased type A natriuretic peptide receptor (NPR-A) expression through a p38 MAPKbeta pathway in inner medullary collecting duct cells. The endothelial and inducible nitric-oxide synthase (eNOS and iNOS respectively) genes are also expressed in this nephron segment and are thought to play a role in regulating urinary sodium concentration. We sought to determine whether changes in tonicity might regulate NOS gene expression, and if so, whether these latter changes might be linked mechanistically to the increase in NPR-A gene expression. Increased extracellular tonicity effected a time-dependent reduction in eNOS and iNOS protein levels, eNOS mRNA levels, and eNOS gene promoter activity over the first 8 h of the incubation. Although levels of the eNOS mRNA and promoter activity had returned to normal after 24 h, eNOS protein levels remained low at 24-36 h, and recovery was not complete even at 48 h. The decrease in eNOS expression was signaled in large part through a p38 MAPK-dependent mechanism. Reduction in eNOS expression together with the concomitant decline in intracellular cyclic GMP levels appears to account for a significant portion of the p38 MAPK-dependent osmotic stimulation of NPR-A gene expression noted previously. Collectively, these findings support the existence of a complex regulatory circuitry in the cells of the inner medullary collecting duct linking two independent cyclic GMP-generating signal transduction systems involved in regulation of urinary sodium concentration.     

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.