Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44±750) in Drosophila Schneider's cells and its puri®-cation to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on¯uorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate speci®city of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identi®ed as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity. Keywords: enzyme glycosylation, glutamate carboxypeptidase II, NAALADase, neuroprotection, zinc metallopeptidase. Membrane glutamate carboxypeptidase II (GCPII) is a metalloproteinase expressed in various tissues and organs (Berger et al. 1995;Chang et al. 1999). Its proteolytic activity was ®rstly recognized in brain cells, where it has been shown to be responsible for the cleavage of N-acetyl-Laspartyl-L-glutamate (NAAG) yielding free glutamate in the extracellular space. Based on the initial characterization of its substrate speci®city, the enzyme was termed N-acetylated alpha-linked-acidic-dipeptidase (NAALADase; Robinson et al. 1987).NAAG is an abundant neuropeptide found in millimolar concentrations in the brain (Coyle 1997). It is a mixed agonist/antagonist at N-methyl-D-aspartate ionotropic receptors and an agonist at the group II metabotropic glutamate receptors (mGlu; Wroblewska et al. 1993Wroblewska et al. , 1997. After release from pre-synaptic terminals, NAAG diffuses from the synaptic cleft and is rapidly hydrolysed by GCPII located on adjacent astrocytes. Free glutamate subsequently acts at the various glutamate receptor subtypes. Excessive receptor activation by glutamate is thought to be at least partially responsible for the neuronal injury caused by stroke (Fagg and Foster 1986). Inhibition of NAAG metabolism was suggested as a possible strategy to attenuate excitatory amino Abbreviations used: Ac, acetyl group; AMC, 7-amino-4-methylcoumarine; DIEA, diisopropylethylamine; DPM, disintegrations per minute; DPP IV, dipeptidyl peptidase IV; FAB-MS, fast atom bombardment mass spectrometry; FBS, foetal bovine serum; Fmoc, 9-...

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10–20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted Nglycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

The nucleotide-binding-domain (NBD)–and leucine-rich repeat (LRR)–containing (NLR) family, pyrin-domain–containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostatespecific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes. Keywords: folate hydrolase, metallopeptidase, molecular modeling, N-acetylated-alpha-linked-acidic dipeptidase II, neurodegeneration, prostate-specific membrane antigen. Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21), also known as N-acetylated-alpha-linked-acidic dipeptidase (NAALADase), is a type-II transmembrane metallopeptidase found in a variety of human tissues, primarily in the central nervous system, small intestine, and prostate (Israeli et al. 1994;Troyer et al. 1995;Silver et al. 1997;Chang et al. 1999;Renneberg et al. 1999;Sokoloff et al. 2000). In the brain, it hydrolyzes the peptide neurotransmitter N-acetyl-Laspartyl-glutamate (NAAG), thus liberating free glutamate (Robinson et al. 1987). Inhibition of GCPII has been shown to be neuroprotective in animal models of stroke, neuropathic pain and other neurodegenerative states (Slusher et al. 1999;Harada et al. 2000;Zhang et al. 2002;Ghadge et al. 2003), and thus it is being considered and tested as a potential therapeutic target (Subasinghe et al. 1990;Jackson et al. 1996;Nan et al. 2000;Whelan 2000;Neale et al. 2005;Tsukamoto et al. 2005;Zhou et al. 2005). In the small intestine, GCPII has been shown to facilitate the absorption Abbreviations used: b-NAAG, N-acetyl-L-aspartyl-b-linked L-glutamate; 2-PMPA, 2-phosphonomethyl-pentanedioic acid; AccQ, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; DPP IV, dipeptidyl peptidase IV; HPLC, high-performance liquid chromatography; KO, knock-out; MD, molecular dynamics; MOPS,propanesulfonic acid; NAAG, N-acetyl-L-aspartyl-L-glutamate; NAALADase, N-acetylated-alpha-li...

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones.Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by E. coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per litre of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner.Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

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