Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.     

Crystal structure of the serine protease domain of prophenoloxidase activating factor-I

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.     

Association of CD38 with nonmuscle myosin heavy chain IIA and Lck is essential for the internalization and activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.     

Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.     

Structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein D

Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8A resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical beta-barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the beta-barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.     

Nucleocytoplasmic shuttling of the zinc finger protein EZI Is mediated by importin-7-dependent nuclear import and CRM1-independent export mechanisms

Nucleocytoplasmic translocation constitutes a foundation for nuclear proteins to exert their proper functions and hence for various biological reactions to occur normally in eukaryotic cells. We reported previously that EZI/Zfp467, a 12 zinc finger motif-containing protein, localizes predominantly in the nucleus, yet the underlying mechanism still remains elusive. Here we constructed a series of mutant forms of EZI and examined their subcellular localization. The results delineated a non-canonical nuclear localization signal in the region covering the 9th to the 12th zinc fingers, which was necessary for nuclear accumulation of EZI as well as sufficient to confer nuclear localizing ability to a heterologous protein. We also found that the N-terminal domain of EZI is necessary for its nuclear export, the process of which was not sensitive to the CRM1 inhibitor leptomycin B. An interaction proteomics approach and the following co-immunoprecipitation experiments identified the nuclear import receptor importin-7 as a molecule that associated with EZI and, importantly, short interfering RNA-mediated knockdown of importin-7 expression completely abrogated nuclear accumulation of EZI. Taken together, these results identify EZI as a novel cargo protein for importin-7 and demonstrate a nucleocytoplasmic shuttling mechanism that is mediated by importin-7-dependent nuclear localization and CRM1-independent nuclear export.     

Effects of fermented oyster mushroom ( Pleurotus ostreats) by-product supplementation on growth performance, blood parameters and meat quality in finishing Berkshire pigs

The objective of the present study was to investigate the effects of fermented oyster mushroom (Pleurotus ostreats) by-production (FOMP) supplementation on the growth performance, blood parameters, carcass traits and meat quality in finishing Berkshire pigs. FOMP was made by mixing oyster mushroom by-production with rice bran and barley bran and this mixture was fermented for 60 days. The experimental diets were 0, 3, 5 and 7% of FOMP added to C, T1, T2 and T3 in the basis diet for 7 weeks. Average daily gain (kg/day) was higher in C and T1 than in T2 and T3 ( P < 0.05). Average daily feed intake (kg/day) and feed conversion increased by the addition of FOMP ( P < 0.05). Total cholesterol and high-density lipoprotein cholesterol were higher in T3 than other treatments ( P < 0.05). Carcass weight (kg) was higher in C and T1 than in T2 and T3 ( P < 0.05). Dressing (%) was higher in C than in T3 ( P < 0.05). Crude protein was lower in T3 than in other treatments ( P < 0.05). Crude fat was higher in T2 and T3 than in C ( P < 0.05). pH₂₄ was higher in C than in other treatments ( P < 0.05). Cooking loss (%) was higher in T1 than T2 ( P < 0.05). Water-holding capacity (%) was higher in C than in T1 ( P < 0.05). In meat colour, CIE a* was lower by the addition of FOMP ( P < 0.05). CIE b* was higher in C than in other treatments ( P < 0.05). In backfat colour, CIE L* was lower in T3 than other treatments ( P < 0.05). CIE b* was lower by addition of FOMP ( P < 0.05). Palmitoleic and oleic acid were higher in T3 than in other treatments ( P < 0.05). Linoleic and arachidonic acids were higher in T2 than in other treatments ( P < 0.05). The results indicate that 3% of FOMP affected the growth performance, carcass traits, meat quality and fatty acid in contrast to addition of 5% of FOMP for Berkshire pigs during the finishing period.     

The impact of plants on the reduction of volatile organic compounds in a small space

This study aims at examining the reduction of indoor air contaminants by plants placed in an indoor space. Field measurements were performed using Aglaonema brevispathum, Pachira aquatica, and Ficus benjamiana, which were verified as air-purifying plants by NASA. Three conditions for the amount of plants and positions were used in two separate rooms whose dimensions are identical. The concentration of Volatile Organic Compounds (VOCs) was monitored three hours after the plants were placed and three days after the plants were placed. The variations of concentration of Benzene, Toluene, Etylbenzene, and Xylene (BTEX), as well as Formaldehyde, which are all known as the major elements of Volatile Organic Compounds were monitored. The amount of reduction in concentration of Toluene and Formaldehyde was monitored 3 hours and 3 days after the plants were placed in the space. The reduction in the concentration of Benzene, Toluene, Etylbenzene, Xylene, and Formaldehyde was significantly greater when plants were present. When plants were placed near a window, the reduction of concentration was greater. The more plants were used, the more a reduction of indoor air contaminants occurred. The effect of reducing the concentration of air contaminants increased when the amount of plants increased, and when the plants were placed in sunny area. The concentration of Toluene was reduced by 45.6 microg/m(3) when 10% of the model space was occupied by Aglaonema brevispathum.