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ObjectiveTo assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA.MethodsAggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies.ResultsThe addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage.ConclusionMatriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.

A study was conducted on the correlation between deletion of the N-terminal region of the AREA protein and a derepressed phenotype with respect to nitrogen metabolite repression. The findings indicate the involvement of the N-terminal region of the AREA protein in mediating nitrogen metabolism. Moreover, the results refute the three-domain model for the structure of the AREA protein.

The activity of the QUTR transcription repressor protein in the quinic acid utilization pathway of Aspergillus nidulans nullifies the action of the QUTA activator protein. Genetic analyses suggest three mechanisms of action of the QUTR protein. The first indicates that high levels of QUTR protein result in the formation of another protein that interacts with QUTA protein to inactivate it. The second possibility is the production of a pool of organic molecules that inactivate QUTA and the third is an inactivating effect through a protein-protein interaction with the QUTA protein.

Previous research has shown that the expression of the eight genes of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans is regulated by a positively acting regulatory protein named QUTA. To better understand the mechanism, the domain structure and function within the QUTA protein is analyzed. Northern blot experiments with messenger RNA support the view that the protein regulates the expression of the qut gene cluster. Western-blot and zinc-binding experiments show that zinc is binded in vitro by a putative zinc binuclear cluster motif within the N-terminus of the protein.

Pre-chorismate shikimate pathway shares two metabolites, 3-dehydroquinate and dehydroshikimate, with the quinate pathway. The enzymes involved in catalyzing the pathway are monofunctional and built around structural modules. The entire quinate pathway is composed by co-option of preformed domains from associated pathways. Domain structure within the AROM protein reveals a dimer of identical subunits with interfacing between two DHQ synthase domains. The pathway can be modulated using principles of metabolic control analysis.

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD ( KD = 2 μM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome. [ABSTRACT FROM AUTHOR]

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered ~11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered ~40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability. [ABSTRACT FROM AUTHOR]

NmrA is a negative transcription-regulating protein that binds to the C-terminal region of the GATA transcription-activating protein AreA. The proposed molecular mechanism of action for NmrA is to inhibit AreA binding to its target promoters. In contrast to this proposal, we report that a C-terminal fragment of AreA can bind individually to GATA-containing DNA and NmrA and that in the presence of a mixture of GATA-containing DNA and NmrA, the AreA fragment binds preferentially to the GATA-containing DNA in vitro. These observations are consistent with NmrA acting by an indirect route, such as by controlling entry into the nucleus. Deletion of the final nine amino acids of a C-terminal fragment of AreA does not affect NmrA binding. Wild-type NmrA binds NAD+(P+) with much greater affinity than NAD(P)H, despite the lack of the consensus GXXGXXG dinucleotide-binding motif. However, introducing the GXXGXXG sequence into the NmrA double mutant N12G/A18G causes an ∼13-fold increase in the KD for NAD+ and a 2.3-fold increase for NADP+. An H37W mutant in NmrA designed to increase the interaction with the adenine ring of NAD+ has a decrease in KD of ∼4.5-fold for NAD+ and a marginal 24% increase for NADP+. The crystal structure of the N12G/A18G mutant protein shows changes in main chain position as well as repositioning of H37, which disrupts contacts with the adenine ring of NAD+, changes which are predicted to reduce the binding affinity for this dinucleotide. The substitutions E193Q/D195N or Q202E/F204Y in the C-terminal domain of NmrA reduced the affinity for a C-terminal fragment of AreA, implying that this region of the protein interacts with AreA. [ABSTRACT FROM AUTHOR]

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-β-ribosyl-4(3H)-pyrimidinone 5′-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase ii catalyzes a distinctive overall reaction from GTP cyclohydrolase 1; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-Å resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II·GMPCPP complex structure shows Arg128 interacting with the α-phosphonate, and thus in the case of GTP, Arg128 is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr105 is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step. [ABSTRACT FROM AUTHOR]