Requirement for hydrophobic Phe residues in Pleurotus ostreatus proteinase A inhibitor 1 for stable inhibition

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) has been shown to be unique among the various serine protease inhibitors in that its C-terminal region appears to be the reactive site responsible for its inhibitory action toward proteases. To investigate in more detail the mechanism of inhibition by POIA1, we have been studying its structural requirements for stable inhibition of proteases. In this study, we focused on hydrophobic Phe residues, which are generally located in the interior of protein molecules. A Phe-->Ala replacement at position 44 or 56 was introduced into a 'parent' mutant of POIA1 that had been converted into a strong and resistant inhibitor of subtilisin BPN' by replacement of its six C-terminal residues with those of the propeptide of subtilisin BPN' and the effects on inhibitory properties and structural stability were examined. Both of the mutated POIA1 molecules not only were found to exhibit decreased ability to bind to subtilisin BPN' (80-fold for the F44A mutant and 13-fold for the F56A mutant), but were also converted to temporary inhibitors that were degraded by the protease. The structural stability of the mutated POIA1 was also lowered, as shown by a 13 degrees C decrease in melting temperature for the F56A mutant. In particular, the F44A mutant was found to lose its tertiary structure, as judged from the circular dichroism spectrum, demonstrating that Phe44 is a strict requirement for structural formation by the POIA1 molecule. These results clearly indicate that stabilization of POIA1 by hydrophobic residues in its molecular interior is required for stable inhibition of the protease. This requirement for a stable tertiary structure is shared with other serine protease inhibitors, but other structural requirements seem to differ, in that strong binding with the protease is required for POIA1 whereas conformational rigidity around the reactive site is essential for many other protease inhibitors.     

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.     

Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.     

Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors

The Rho family GTPase has been implicated in plexin-B1, a receptor for Semaphorin 4D (Sema4D), mediating signal transduction. Rho may also play a function in this signaling pathway as well as Rac, but the mechanisms for Rho regulation are poorly understood. In this study, we have identified two kinds of PDZ domain-containing Rho-specific guanine nucleotide exchange factors (RhoGEFs) as proteins interacting with plexin-B1 cytoplasmic domain. These PDZ domain-containing RhoGEFs showed significant homology to human KIAA0380 (PDZ-RhoGEF) and LARG (KIAA0382), respectively. Both KIAA0380 and LARG could bind plexin-B1 and a deletion mutant analysis of plexin-B1, KIAA0380 and LARG revealed that KIAA0380 and LARG bound plexin-B1 cytoplasmic tail through their PDZ domains. The tissue distribution analysis indicated that plexin-B1 was co-localized with KIAA0380 and LARG in various tissues. Immunocytochemical analysis showed that LARG was recruited to plasma membrane by plexin-B1. These results suggest that PDZ domain-containing RhoGEFs play a role in Sema4D-plexin-B1 mediating signal transduction.     

Arg tyrosine kinase is involved in homologous recombinational DNA repair

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.     

Active-dimeric form of lipoprotein lipase increases in the adipose tissue of patients with rheumatoid arthritis treated with prednisolone

A thermodynamic analysis of the interaction of 125I-labeled human chorionic gonadotropin (IhCG) with two of its monoclonal antibodies (MAbs) was carried out. The dissociation profile of IhCG-MAb complex conforms to a two-step model. vant Hoff enthalpies were calculated with the K(A) (equilibrium constant) values obtained from dissociation at different temperatures. Free energy and entropy changes were calculated using the standard equations. DeltaH values for one of the MAbs, viz. VM7 were favorable at temperatures beyond 30 degrees C. Interestingly, the DeltaS values were also favorable at all temperatures. In the case of MAb VM4a, however, the interaction throughout the temperature range was driven by large favorable entropic contributions, indicating the importance of hydrophobic interaction in the binding of this MAb to hCG. The energetics of the interaction of these two monoclonals with hCG is discussed.     

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.