Volume 389, Issue 2

Tissue kallikrein (kallikrein 1) was first identified in pancreas and is the namesake of the kallikrein-related peptidase (KLK) family. KLK1 and the other 14 members of the human KLK family are encoded by 15 serine protease genes clustered at chromosome 19q13.4. Our Northern blot analysis of 19 normal human tissues for expression of KLK4 to KLK15 identified pancreas as a common expression site for the gene cluster spanning KLK5 to KLK13 , as well as for KLK15 which is located adjacent to KLK1 . Consistent with previous reports detailing the ability of KLK genes to generate organ- and disease-specific transcripts, detailed molecular and in silico analyses indicated that KLK5 and KLK7 generate transcripts in pancreas variant from those in skin or ovary. Consistently, we identified in the promoters of these KLK genes motifs which conform with consensus binding sites for transcription factors conferring pancreatic expression. In addition, immunohistochemical analysis revealed predominant localisation of KLK5 and KLK7 in acinar cells of the exocrine pancreas, suggesting roles for these enzymes in digestion. Our data also support expression patterns derived from gene duplication events in the human KLK cluster. These findings suggest that, in addition to KLK1, other related KLK enzymes will function in the exocrine pancreas.

Polyamines are essential polycationic molecules involved in multiple cellular events, including cell differentiation, division and death. Inhibition of polyamine biosynthesis has been considered in diverse therapeutic strategies ranging from tumour suppressors to anti-parasitic agents. In the human malaria parasite, Plasmodium falciparum , inhibition of ornithine decarboxylase (ODC) results in the arrest of schizogony due to polyamine depletion. However, the exact physiological role of the polyamines in the parasite is unknown. Here, we present results of the depletion of polyamines in the malaria parasite by α-difluoromethylornithine inhibition of ODC, as observed with differential transcriptome profiling. Upon depletion of their endogenous polyamines, the up- and downregulated parasite transcripts were selected with suppression subtractive hybridisation and differences were detected using blots or DNA microarrays. A direct linkage between polyamine depletion and the differential expression of two distinct transcripts was observed, indicating the existence of a transcriptional feedback response in the P. falciparum transcriptome upon drug challenge. The data presented provide input into the role of the polyamines in the cellular biology of P. falciparum and contribute towards the validation of polyamine biosynthesis as an antimalarial target.

Bacterial thiM riboswitches contain aptamer domains that bind the metabolite thiamine pyrophosphate (TPP). Binding of TPP to the aptamer domain induces structural rearrangements that are relayed to the expression domain, thereby interfering with gene expression. Here, we report identification of three putative thiM riboswitches from different bacteria and analysis of their secondary structures. Chemical probing revealed that the riboswitches share similar secondary structures in their aptamer domains that can communicate with the highly variant expression domains in a mechanism likely involving sequestration of the Shine-Dalgarno sequence. Remarkably, the aptamer domain of the thiM gene of Desulfovibrio vulgaris binds TPP with similar affinity and selectivity as that of Escherichia coli , although nucleotides previously shown to form direct contacts to the metabolite are mutated. We also designed small RNA hairpins for each riboswitch that bind the RNA only in the absence of the metabolite. Our study shows that aptamer domains in riboswitches with high similarity in their secondary structures can communicate with a broad variety of non-related expression domains by similar mechanisms.

Angiogenesis plays an important role in normal physiology of blood vessel growth, but can contribute to the pathogenesis of diseases, such as cancer. A new anti-angiogenic recombinant kringle protein, composed of the fused domains of human apolipoprotein(a) carboxyl-terminal kringle IV-10 and kringle V, was expressed in Pichia pastoris and human colorectal carcinoma (HCT 116) cells to investigate its influence on angiogenesis and tumor growth. The mature recombinant protein exhibited the characteristic features of kringle-containing proteins (glycosylation and disulfide bond formation) and, when added to cultures of human umbilical vein endothelial cell, resulted in a 31% decrease in proliferation relative to untreated controls ( p <0.05). The neo-angiogenesis was diminished by 63% in chick embryos treated with 10 μg recombinant protein compared with 7% for phosphate buffer solution-treated embryos ( p <0.01). Transfection of a kringle IV-10-kringle V fusion protein construct into HCT 116 cells decreased tumorigenesis and inhibited tumor growth in vivo without affecting tumor cell proliferation. HCT 116 cells that expressed recombinant protein displayed a much lower relative growth ratio of 8% ( p <0.01) against the control tumor cells. From these results, we conclude that human apolipoprotein(a) carboxyl-terminal kringle IV-10-kringle V fusion protein is an effective inhibitor of angiogenesis and angiogenesis-dependent tumor growth.

Pseudin-2 is a cationic α-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10 -9 –10 -6   m , pseudin-2, and its [Lys 18 ], [Phe 8 ], and [ d -Lys 3 , d -Lys 10 , d -Lys 14 ] derivatives, stimulated insulin release from the BRIN-BD11 clonal β-cell line without increasing release of lactate dehydrogenase. The [Lys 18 ] analogue was the most potent (46% increase in insulin release at 10 -9   m ) and the most effective (215% increase in insulin release at 10 -6   m ). The more cationic [Lys 3 ,Lys 10 ,Lys 14 ] and [Lys 3 ,Lys 10 ,Lys 14 ,Lys 21 ] analogues lacked insulinotropic action and the more hydrophobic [Phe 16 ] analogue was cytotoxic at concentrations ≥10 -7   m . Pseudin-2 and [Lys 18 ]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys 18 ]-pseudin-2 (10 -8   m ) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca 2+ -independent pathways and identify [Lys 18 ]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.

Structures of mammalian carboxylesterases (CEs) reveal the presence of a ‘side door’ that is proposed to act as an alternative pore for the trafficking of substrates and products. p -Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific ‘gate’ residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower k cat and k cat / K m values. The mutation with the most significant impact on catalysis was the L362R mutant ( k cat / K m was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.

Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr 318 at the active site of this enzyme. The substitution of Tyr 318 to Phe decreased k cat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.

Efforts were made to develop a human adipocyte model that is useful for toxicological studies in vitro . For this purpose, a stem cell line derived from human bone marrow cells, originally from an adult, was induced to differentiate towards adipocytes by treating them with insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine for 3 d, followed by additional incubation for 3 d in Dulbecco's modified Eagle's medium supplemented with insulin only. In most cases, thus differentiated cells through such one cycle of differentiation treatment were further subjected to the second cycle of differentiation. The resulting 2-cycle differentiated cells were found to exhibit many characteristics of typical adipocytes. Dioxin (TCDD), when added at the beginning of their treatment with differentiation-inducing hormone cocktail, clearly prevented them from becoming adipocytes, as in the case of TCDD-treated 3T3-L1 cells. Furthermore, TCDD, even when administered to previously differentiated human mesenchymal stem cells (hMSC) adipocytes, consistently induced the sign of inflammatory responses during the early period of TCDD action (24 h), which was followed by gradual loss of adipocyte-specific markers during the 5-d incubation period. In conclusion, hMSC-derived adipocytes appear to offer a promising human cell model suited for future toxicological studies.

Few genes are known to be involved in renal cell carcinoma (RCC) development and progression. The cell-specific transcription factor hepatocyte nuclear factor 4α (HNF4α) is down-regulated in RCC and we have shown that HNF4α inhibits cell proliferation in the embryonic kidney cell line HEK293. To clarify the possible tumor suppressor activity of HNF4α we analyzed the whole human expression profile in HEK293 cells upon HNF4α induction. By comparing induced and uninduced cells, we identified 1411 differentially expressed genes. Using RNA interference, we screened 56 HNF4α-regulated genes for their possible role in mediating inhibition of cell proliferation triggered by HNF4α. We demonstrate that 14 of these regulated genes are able to contribute to the inhibitory effect of HNF4α on cell proliferation, including well-known cancer genes, such as CDKN1A (p21), TGFA, MME (NEP) and ADAMTS1. In addition, the genes SEPP1, THEM2, BPHL, DSC2, ANK3, ALDH6A1, EPHX2, NELL2, EFHD1 and PROS1 are also part of the network of HNF4α target genes that regulate proliferation in HEK293 cells. Therefore, we postulate that HNF4α orchestrates, at least, these 14 genes to regulate cell proliferation in HEK293 cells and that down-regulation of HNF4α could contribute to the progression of kidney cancer.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine K-cells in response to nutrient absorption. This study has utilised numerous well-characterised dipeptidyl peptidase IV-resistant GIP analogues to evaluate the glucagonotropic actions of GIP in Wistar rats and isolated rat islets. Intraperitoneal administration of GIP analogues (25 nmol/kg body weight) in combination with glucose had no effect on circulating glucagon concentrations compared to controls in Wistar rats. However, plasma glucose concentrations were significantly ( p <0.05 to p <0.001) lowered by the GIP-receptor agonists, N -AcGIP, GIP(Lys 37 )PAL and N -AcGIP(Lys 37 )PAL. The GIP antagonist, (Pro 3 )GIP, caused a significant ( p <0.05) reduction in glucagon levels following concurrent administration with saline in Wistar rats. In isolated rat islets native GIP induced a significant ( p <0.01) enhancement of glucagon release at basal glucose concentrations, which was completely annulled by (Pro 3 )GIP. Furthermore, glucagon release in the presence of GLP-1, GIP(Lys 37 )PAL, N -AcGIP(Lys 37 )PAL and (Pro 3 )GIP was significantly ( p <0.05 to p <0.001) decreased compared to native GIP in isolated rat islets. These data indicate a modest effect of GIP on glucagon secretion from isolated rat islets, which was not observed in vivo . However, the GIP agonists N -AcGIP, GIP(Lys 37 )PAL and N -AcGIP(Lys 37 )PAL had no effect on glucagon release demonstrating an improved therapeutic potential for the treatment of type 2 diabetes.

Cathepsin V is a lysosomal cysteine peptidase highly expressed in corneal epithelium; however, its function in the eye is still unknown. Here, we describe the capability of cathepsin V to hydrolyze plasminogen, which is also expressed in human cornea at levels high enough to produce physiologically relevant amounts of angiostatin-related molecules. The co-localization of these two proteins suggests an important role for the enzyme in the maintenance of corneal avascularity, essential for optimal visual performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of plasminogen digestion by cathepsin V revealed the generation of three major products of 60, 50 and 40 kDa, which were electrotransferred to polyvinylidene difluoride membranes and excised for characterization. NH 2 -terminal amino acid sequencing of these fragments revealed the sequences EKKVYL, TEQLAP and LLPNVE, respectively. These data are compatible with cleavage sites at plasminogen F94–E95, S358–T359 and V468–L469 peptide bonds generating fragments of the five-kringle domains. In contrast, we did not detect any plasminogen degradation by cathepsins B, K and L. Using a Matrigel assay, we confirmed the angiogenesis inhibition activity on endothelial cells caused by plasminogen processing by cathepsin V. Our results suggest a novel physiological role for cathepsin V related to the control of neovascularization in cornea.