Expression of RARRES1 and AGBL2 and progression of conventional renal cell carcinoma.

BACKGROUND: Approximately 15% of clinically localised conventional renal cell carcinoma (RCC) will develop metastasis within 5 years of follow-up. The aim of this study was to identify biomarkers predicting the postoperative tumour relapse. METHODS: Tissue microarrays of conventional RCC from a cohort of 691 patients without metastasis at the time of operation were analysed by immunohistochemistry for the expression of carboxypeptase inhibitor RARRES1 and its substrate carboxypeptidase AGBL2. Univariate and multivariate Cox regression models were addressed to postoperative tumour relapse and the metastasis-free survival time was estimated by Kaplan-Meier analysis. RESULTS: In multivariate analysis, the lack of staining or cytoplasmic staining of RARRES1 was a significant risk factor indicating five times higher risk of cancer relapse. Combining its co-expression with AGBL2, we found that RARRES1 cytoplasmic/negative and AGBL2-positive/negative staining is a significant risk factor for tumour progression indicating 11-15 times higher risk of cancer relapse, whereas the membranous RARRES1 expression, especially its co-expression with AGBL2, associated with excellent disease outcome. CONCLUSIONS: RARRES1 and AGBL2 expression defines groups of patients at low and high risk of tumour progression and may direct an active surveillance to detect metastasis as early as possible and to apply adjuvant therapy.

Overexpression of CPXM2 predicts an unfavorable prognosis and promotes the proliferation and migration of gastric cancer.

Carboxypeptidase X, M14 family member 2 (CPXM2), has been associated with several human disorders such as developmental diseases. However, whether CPXM2 is involved in oncogenesis or tumor progression remains unclear. In the present study, we used clinical samples from gastric cancer (GC) patients to investigate potential roles of CPXM2 in GC. We also analyzed datasets from the Oncomine database, The Cancer Genome Atlas (TCGA), and the Kaplan­Meier Plotter to validate these results. We found that CPXM2 was overexpressed in GC and that the overexpression was associated with an unfavorable prognosis, regardless of the Lauren classification and tumor node metastasis staging. In addition, knockdown of CPXM2 in cultured GC cells significantly impeded cell proliferation and migration, as indicated by the cholecystokinin octapeptide, colony formation assay, scratch wound healing assay, and Transwell® migration assay. Furthermore, gene set enrichment analysis using RNA­seq data from TCGA indicated that high CPXM2 expression in GC patients was positively correlated with the HALLMARK_APICAL_JUNCTION and HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION gene sets. Finally, western blotting results revealed that several key molecules involved in the epithelial mesenchymal transition were regulated by CPXM2. Taken together, these results imply an active role for CPXM2 in promoting tumor aggressiveness via epithelial to mesenchymal transition (EMT) modulation in GCs.

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.

Deficient prolylcarboxypeptidase gene and protein expression in left ventricles of spontaneously hypertensive rats (SHR).

Prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine peptidase that inactivates angiotensin II and activates pre-kallikrein, is thought to have anti-hypertensive and anti-proliferative roles in cardiovascular homeostasis. We hypothesized that PRCP function may be altered in heart tissue under conditions that predispose to left ventricle hypertrophy (LVH) in rats. We therefore used real-time PCR and western-blotting to examine the mRNA and protein expression of PRCP in the hearts of spontaneously hypertensive rats (SHR) at pre-hypertensive (5-week-old) and hypertensive (16-week-old) stages compared with age-matched hypertensive (2 kidney-1 clip; 2K-1C) rats and normotensive Wistar rats. PRCP mRNA expression was significantly reduced in hearts of 5- and 16-week-old SHR compared with age-matched Wistar controls, 2K-1C hypertensive rats and sham-operated normotensive rats. There were no significant differences in the PRCP mRNA and protein expression levels in hearts from hypertensive renovascular and sham-operated normotensive rats. Prolonged treatment of SHR with the AT1 receptor antagonist losartan (40 mg/kg, gavage for 8 weeks) reduced the left ventricular weight/body weight ratio (LVW/BW), as well as the mRNA expression of collagen type 1, collagen type 3 and MMP9 in left ventricular tissue, without affecting PRCP gene and protein expression. Our results suggest that diminished PRCP gene and protein expression might be constitutionally involved in the SHR phenotype. In addition, since neither the development of arterial hypertension in the 2K-1C model nor its successful treatment in SHR altered PRCP gene and protein expression in heart tissue, it appears unlikely that PRCP function is regulated by the renin-angiotensin system or by afterload conditions.

[Expression of the thermostable carboxypeptidase Taq gene in Pichia pastoris GS115].

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.

Zebrafish cytosolic carboxypeptidases 1 and 5 are essential for embryonic development.

The cytosolic carboxypeptidases (CCPs) are a subfamily of metalloenzymes within the larger M14 family of carboxypeptidases that have been implicated in the post-translational modification of tubulin. It has been suggested that at least four of the six mammalian CCPs function as tubulin deglutamylases. However, it is not yet clear whether these enzymes play redundant or unique roles within the cell. To address this question, genes encoding CCPs were identified in the zebrafish genome. Analysis by quantitative polymerase chain reaction indicated that CCP1, CCP2, CCP5, and CCP6 mRNAs were detectable between 2 h and 8 days postfertilization with highest levels 5-8 days postfertilization. CCP1, CCP2, and CCP5 mRNAs were predominantly expressed in tissues such as the brain, olfactory placodes, and pronephric ducts. Morpholino oligonucleotide-mediated knockdown of CCP1 and CCP5 mRNA resulted in a common phenotype including ventral body curvature and hydrocephalus. Confocal microscopy of morphant zebrafish revealed olfactory placodes with defective morphology as well as pronephric ducts with increased polyglutamylation. These data suggest that CCP1 and CCP5 play important roles in developmental processes, particularly the development and functioning of cilia. The robust and similar defects upon knockdown suggest that each CCP may have a function in microtubule modification and ciliary function and that other CCPs are not able to compensate for the loss of one.

Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: differences and similarities with its orthologue in Trypanosoma cruzi.

Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co(2+) inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns.

Structure of C3PO and mechanism of human RISC activation.

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide-passenger) siRNA onto Argonaute (Ago2) and removing the passenger strand. Ago2 contributes critically to RISC activation by nicking the passenger strand. Here we reconstituted duplex siRNA-initiated RISC activity using recombinant human Ago2 (hAgo2) and C3PO, indicating that C3PO has a critical role in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as an endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation, in which Ago2 directly binds duplex siRNA and nicks the passenger strand, and then C3PO activates RISC by degrading the Ago2-nicked passenger strand.

Effect of collagen I and aortic carboxypeptidase-like protein on 3T3-L1 adipocyte differentiation.

Aortic carboxypeptidase-like protein (ACLP) is a secreted protein expressed in preadipocytes and down-regulated during adipogenesis. Results from previous studies on the influence of ACLP overexpression on adipogenesis vary from no effect to complete inhibition. We hypothesized that ACLP may modulate adipogenesis in the presence of collagen I, a protein to which it binds. We compared control (pLXSN) 3T3-L1 preadipocytes with 3T3-L1 preadipocytes stably overexpressing ACLP (pLXSN-ACLP) that were grown in standard vs collagen I-coated dishes. Aortic carboxypeptidase-like protein overexpression, via retroviral transduction, resulted in a 3.2-fold increase in ACLP cellular levels and a 2.1-fold increase in ACLP levels released into medium. Aortic carboxypeptidase-like protein overexpression did not inhibit differentiation in standard dishes. In collagen I-coated dishes compared with standard dishes, control preadipocytes, when induced to differentiate, exhibited the same increase in triacylglycerol accumulation, but showed a significantly higher induction of fatty acid synthase (1.6-fold more), peroxisome proliferator-activated receptor γ (1.4-fold more), and CCAAT/enhancer-binding protein α (1.4-fold more). Aortic carboxypeptidase-like protein overexpression significantly reduced this enhanced induction of fatty acid synthase, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α by 65%, 59%, and 66%, respectively, but had no effect on the accumulation of triacylglycerol during differentiation. Finally, studies on proadipogenic insulin signaling in ACLP-overexpressing preadipocytes demonstrated that insulin-stimulated Akt phosphorylation was significantly decreased by 27% in cells cultured in collagen I-coated dishes vs standard dishes. Our data suggest that ACLP inhibits certain aspects of 3T3-L1 adipogenesis in a collagen I-rich environment.

Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH.

[Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody].

Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.

Efficient bacterial expression of fusion proteins and their selective processing by a recombinant Kex-1 protease.

A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

[Preparation and characterization of rabbit antibody against human mast cell carboxypeptidase].

AIM: To prepare antibody against human mast cell carboxypeptidase (hMC-CP) by using recombinant hMC-CP expressed in E.coli, and to characterize the antibody. METHODS: hMC-CP was expressed in E.coli with L-Arabinose induction and purified through Ni-NTA column. The purified hMC-CP as immunogen was used to immunize rabbit. The titer and the specificity of the rabbit anti-hMC-CP antibody was analyzed by indirect ELISA and Western blot respectively. RESULTS: The hMC-CP was successfully expressed and purified. The polyconal anit-hMC-CP antibody was prepared by immunizing rabbit using the purified recombinant protein, The titer of the generated antiserum was 1:6 400 by ELISA. Western blot analysis showed that this antibody could bind with hMC-CP specifically. CONCLUSION: The rabbit anti-hMC-CP antibody with high titer and high specificity has been prepared by using purified recombinant hMC-CP as immunogen, which lays the foundation for further research on detection and function of hMC-CP.

Carboxypeptidase D: a novel TGF-beta target gene dysregulated in patients with lupus erythematosus.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that mainly acts as an inhibitor of immune functions. A lack of functional TGF-beta leads to autoimmune disease in animal models and dysregulated TGF-beta signaling is implicated in human autoimmune diseases. To define target genes that play a part in the inhibitory role of TGF-beta in the immune system, we have identified genes stimulated by TGF-beta in macrophages by gene-chip analysis. One of the TGF-beta regulated genes is carboxypeptidase D (CpD), a 180-kDa type I membrane protein. We have demonstrated that CpD is regulated by TGF-beta in various cell types of both, murine and human origin and, interestingly, is significantly downregulated in CD14 positive cells isolated from patients with lupus erythematosus (LE). Moreover, we show that downregulation of CpD leads to downmodulation of TGF-beta itself, suggesting a role for CpD in a positive feedback loop, providing further evidence for a role of this enzyme in LE. To our knowledge, this is the first report that demonstrates carboxypeptidase D as a TGF-beta target gene that is implicated in the pathogenesis of LE.

Tissue expression of the novel serine carboxypeptidase Scpep1.

We previously identified a novel gene designated retinoid-inducible serine carboxypeptidase (RISC or Scpep1). Here we characterize a polyclonal antibody raised to Scpep1 and assess its localization in mouse cells and tissues. Western blot analysis revealed an immunospecific approximately 35-kDa protein corresponding to endogenous Scpep1. This protein is smaller than the predicted approximately 51-kDa, suggesting that Scpep1 is proteolytically cleaved to a mature enzyme. Immunohistochemical studies demonstrate Scpep1 expression in embryonic heart and vasculature as well as in adult aortic smooth muscle cells and endothelial cells. Scpep1 displays a broad expression pattern in adult tissues with detectable levels in epithelia of digestive tract and urinary bladder, islet of Langerhans, type II alveolar cells and macrophages of lung, macrophage-like cells of lymph nodes and spleen, Leydig cells of testis, and nerve fibers in brain and ganglia. Consistent with previous mRNA studies in kidney, Scpep1 protein is restricted to proximal convoluted tubular epithelium (PCT). Immunoelectron microscopy shows enriched Scpep1 within lysosomes of the PCT, and immunofluorescence microscopy colocalizes Scpep1 with lysosomal-associated membrane protein-2. These results suggest that Scpep1 is a widely distributed lysosomal protease requiring proteolytic cleavage for activity. The highly specific Scpep1 antibody characterized herein provides a necessary reagent for elucidating Scpep1 function.

The role of AEBP1 in sex-specific diet-induced obesity.

Obesity is an important risk factor for heart disease, diabetes, and certain cancers, but the molecular basis for obesity is poorly understood. The transcriptional repressor AEBP1, which functions as a negative regulator of PTEN through a protein-protein interaction, is highly expressed in the stromal compartment of adipose tissues, including proliferative preadipocytes, and its expression is abolished in terminally differentiated, nonproliferative adipocytes. Here we show that transgenic overexpression of AEBP1 during adipogenesis coupled with a high-fat diet (HFD) resulted in massive obesity in female transgenic (AEBP1(TG)) mice via adipocyte hyperplasia. AEBP1 levels dynamically changed with aging, and HFD induced AEBP1 expression in females. Thus, HFD-fed AEBP1(TG) females display hyperinduction of AEBP1 and a marked reduction of PTEN level with concomitant hyperactivation of the survival signal in white adipose tissue. Our results suggest that AEBP1 plays a key functional role in in vivo modulation of adiposity via fat-cell proliferation and is involved in a sex-specific susceptibility to diet-induced obesity by the estrogen signaling pathway.

Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats.

Angiotensin converting enzyme 2 (ACE2), a newly discovered member of the renin-angiotensin system (RAS), is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides from angiotensin II. The aim of the present study was to evaluate whether overexpression of ACE2 could protect the heart from angiotensin II-induced hypertrophy and fibrosis. Lentiviral vector encoding mouse ACE2 (lenti-mACE2) or GFP was injected intracardially in 5-day-old Sprague-Dawley rats. This resulted in expression of mACE2 in cardiac tissue for the duration of the study. Infusion of 200 ng kg-1 min-1 angiotensin II for 4 weeks resulted in an 80 mmHg increase in systolic blood pressure, a significant increase in the heart weight to body weight ratio (HW:BW), and marked myocardial fibrosis in control rats. Transduction with lenti-mACE2 resulted in significant attenuation of the increased HW:BW and myocardial fibrosis induced by angiotensin II infusion. These observations demonstrate that ACE2 overexpression results in protective effects on angiotensin II-induced cardiac hypertrophy and fibrosis.

Myocardial infarction increases ACE2 expression in rat and humans.

AIMS: Angiotensin converting enzyme (ACE) 2 catalyses the cleavage of angiotensin (Ang) I to Ang 1-9 and of Ang II to Ang 1-7. ACE2 deficiency impairs cardiac contractility and upregulates hypoxia-induced genes, suggesting a link with myocardial ischaemia. We studied the expression of ACE2 after myocardial infarction (MI) in the rat as well as in human failing hearts. METHODS AND RESULTS: Rats were killed at days 1, 3, and 28 after MI, or treated for 4 weeks with the ACE inhibitor ramipril (1 mg/kg). Cardiac gene and protein expression of ACE and ACE2 were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry/activity assays/in vitro autoradiography, respectively. Both ACE (P = 0.022) and ACE2 (P = 0.015) mRNA increased in the border/infarct area compared with the viable area at day 3 after MI. By day 28, increases in ACE (P = 0.005) and ACE2 (P = 0.006) mRNA were also seen in the viable myocardium of MI rats compared with myocardium of control rats. ACE2 protein localized to macrophages, vascular endothelium, smooth muscle, and myocytes. Ramipril attenuated cardiac hypertrophy and inhibited cardiac ACE. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in all areas of the MI rat heart. Immunoreactivity of both ACE and ACE2 increased in failing human hearts. CONCLUSION: The increase in ACE2 after MI suggests that it plays an important role in the negative modulation of the renin angiotensin system in the generation and degradation of angiotensin peptides after cardiac injury.

Persistent infection of SARS coronavirus in colonic cells in vitro.

Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.

Increased ACE 2 and decreased ACE protein in renal tubules from diabetic mice: a renoprotective combination?

Unlike the ubiquitous angiotensin-converting enzyme (ACE), the ACE-related carboxypeptidase 2 (ACE 2) is predominantly expressed in the heart, kidney, and testis. ACE 2 degrades angiotensin (Ang) II to Ang (1-7) and Ang I to Ang (1-9). We investigated the expression of ACE and ACE 2 in a rodent model of type 2 diabetes. ACE and ACE 2 were measured in kidney and heart from 8-week-old no diabetic control (db/m) mice and diabetic (db/db) mice, which at this young age have obesity and hyperglycemia without nephropathy. In renal cortical tissue, ACE mRNA was reduced (db/db 0.31+/-0.06 versus db/m 0.99+/-0.05; P<0.005), whereas ACE 2 mRNA was not (db/db 0.94+/-0.05 versus db/m 1.03+/-0.11, NS). ACE protein was markedly reduced in kidney cortex of db/db mice (db/db 0.24+/-0.13 versus db/m 1.02+/-0.12; P<0.005), and this was associated with a corresponding decrease in renal ACE activity (db/db 12.7+/-3.7 versus db/m 61.6+/-4.4 mIU/mg protein; P<0.001). ACE 2 protein, by contrast, was increased in kidneys from diabetic mice (db/db 1.39+/-0.14 versus db/m 0.53+/-0.04; P<0.005). An increase in ACE 2 protein and a decrease in ACE protein, respectively, were also seen by immunostaining of renal cortical tubules from the db/db mice. In heart tissue, there were no significant differences between db/db and db/m mice in either ACE mRNA and protein or ACE 2 mRNA and protein. We conclude that in young db/db mice, ACE 2 protein in renal cortical tubules is increased, whereas ACE protein is decreased. We propose that the pattern of low ACE protein coupled with increased ACE 2 protein expression may be renoprotective in early stages of diabetes.