Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg(26)-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-A resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel beta-sheet wrapped around a five-turn alpha-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes.     

Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.     

Extracellular matrix proteoglycans control the fate of bone marrow stromal cells

Extracellular matrix glycoproteins and proteoglycans bind a variety of growth factors and cytokines thereby regulating matrix assembly as well as bone formation. However, little is known about the mechanisms by which extracellular matrix molecules modulate osteogenic stem cells and bone formation. Using mice deficient in two members of the small leucine-rich proteoglycans, biglycan and decorin, we uncovered a role for these two extracellular matrix proteoglycans in modulating bone formation from bone marrow stromal cells. Our studies showed that the absence of the critical transforming growth factor-beta (TGF-beta)-binding proteoglycans, biglycan and decorin, prevents TGF-beta from proper sequestration within the extracellular matrix. The excess TGF-beta directly binds to its receptors on bone marrow stromal cells and overactivates its signaling transduction pathway. Overall, the predominant effect of the increased TGF-beta signaling in bgn/dcn-deficient bone marrow stromal cells is a "switch in fate" from growth to apoptosis, leading to decreased numbers of osteoprogenitor cells and subsequently reduced bone formation. Thus, biglycan and decorin appear to be essential for maintaining an appropriate number of mature osteoblasts by modulating the proliferation and survival of bone marrow stromal cells. These findings underscore the importance of the micro-environment in controlling the fate of adult stem cells and reveal a novel cellular and molecular basis for the physiological and pathological control of bone mass.     

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.     

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.     

The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila

The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.     

Kinetic and structural properties of disulfide engineered phospholipase A2: insight into the role of disulfide bonding patterns

The family of secreted 14 kDa phospholipase A(2) (PLA2) enzymes have a common motif for the catalytic site but differ in their disulfide architecture. The functional significance of such structural changes has been analyzed by comparing the kinetic and spectroscopic properties of a series of disulfide mutants engineered into the sequence of pig pancreatic IB PLA2 to resemble the mammalian paralogues of the PLA2 family [Janssen et al. (1999) Eur. J. Biochem. 261, 197-207, 1999]. We report a detailed comparison of the functional parameters of pig iso-PLA2, as well as several of the human homologues, with these disulfide engineered mutants of pig IB PLA2. The crystal structure of the ligand free and the active site inhibitor-MJ33 bound forms of PLA2 engineered to have the disulfide bonding pattern of group-X (eng-X) are also reported and compared with the structure of group-IB and human group-X PLA2. The engineered mutants show noticeable functional differences that are rationalized in terms of spectroscopic properties and the differences detected in the crystal structure of eng-X. A major difference between the eng-mutants is in the calcium binding to the enzyme in the aqueous phase, which also influences the binding of the active site directed ligands. We suggest that the disulfide architecture of the PLA2 paralogues has a marginal influence on interface binding. In this comparison, the modest differences observed in the interfacial kinetics are attributed to the changes in the side chain residues. This in turn influences the coupling of the catalytic cycle to the calcium binding and the interfacial binding event.     

Highly specific expression of luciferase gene in lungs of naïve nude mice directed by prostate-specific antigen promoter

PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.