Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis.     

Membrane trafficking in plants: new discoveries and approaches

The general organization and function of the endomembrane system is highly conserved in eukaryotic cells. In addition, increasing numbers of studies demonstrate that normal plant growth and development are dependent on specialized tissue and subcellular-specific components of the plant membrane trafficking machinery. New approaches, including chemical genomics and proteomics, will likely accelerate our understanding of the diverse functions of the plant endomembrane system.     

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.     

Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay

We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

The pi-helix translates structure into function

A search for the occurrence of the rare pi-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the pi-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight pi-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the pi-helix has evolved to provide unique structural features within a variety of proteins.     

CPR-Total (TAFI and activated TAFI) levels in plasma/serum of hemophiliacs

Arginine carboxypeptidase (CPR) is a single-chain plasma protein generated during coagulation from a precursor (proCPR). proCPR is the same molecule as thrombin activable fibrinolysis inhibitor (TAFI), which retards fibrin clot lysis in vitro and most likely modulates fibrinolysis in vivo. In this study, the amount of CPR-total, which includes proCPR (TAFI) and CPR (activated TAFI), in hemophiliac patients was evaluated using a newly developed enzyme linked immunosorbent assay (ELISA). The amount of CPR-total in plasma or serum of most of the hemophiliac patients was in the range of healthy individuals. There was no significant difference in hemophiliac patients with or without HIV-1 infection. However, two out of the 74 hemophiliac patients showed a significantly high level. The upregulation of CPR-total might contribute to compensate for inefficient coagulation in some hemophiliac individuals.     

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY∗ is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY∗, which carries a mutation homologous to yeast CPY∗, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY∗-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY∗-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY∗-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.     

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.