Angiotensin I-Converting Enzyme Mutation (Trp1197Stop) Causes a Dramatic Increase in Blood ACE

BACKGROUND:Angiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots. METHODOLOGY/PRINCIPAL FINDINGS:We have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension. CONCLUSIONS/SIGNIFICANCE:We have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease

Angiotensin I-Converting Enzyme Gln1069Arg Mutation Impairs Trafficking to the Cell Surface Resulting in Selective Denaturation of the C-Domain

Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death.Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10–20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold.Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine

A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP

BACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S 1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE--between Arg1203 and Ser1204--was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another.The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase)

Neutrophils in animal models of autoimmune disease

Neutrophils have traditionally been thought to play only a peripheral role in the genesis of many autoimmune and inflammatory diseases. However, recent studies in a variety of animal models suggest that these cells are central to the initiation and propagation of autoimmunity. The use of mouse models, which allow either deletion of neutrophils or the targeting of specific neutrophil functions, has revealed the many complex ways these cells contribute to autoimmune/inflammatory processes. This includes generation of self antigens through the process of NETosis, regulation of T-cell and dendritic cell activation, production of cytokines such as BAFF that stimulate self-reactive B-cells, as well as indirect effects on epithelial cell stability. In comparing the many different autoimmune models in which neutrophils have been examined, a number of common underlying themes emerge - such as a role for neutrophils in stimulating vascular permeability in arthritis, encephalitis and colitis. The use of animal models has also stimulated the development of new therapeutics that target neutrophil functions, such as NETosis, that may prove beneficial in human disease. This review will summarize neutrophil contributions in a number of murine autoimmune/inflammatory disease models. © 2016 Elsevier Ltd

The development of Nemurella pictetii KLAPÁLEK (Plecoptera: Nemouridae) in two springstreams in central Europe

From April 1997 to June 1998 Nemurella pictetii populations were regularly sampled in two springstreams at 220 and 850 m a.s.l., respectively, in Hesse (Germany), at approximately 51°N. Random samples of larvae were taken at three week intervals during the vegetation period, and once a month during winter. Sex, instar, body length, head capsule width and wing pad lenght of all larvae were recorded. Temperatures were recorded every hour, temporal patterns of temperature agreed closely between sites. Mean winter lows were 3.9 °C at both sites, the mean summer high was 11.9 °C at the lower site, as opposed to 9.6 °C at the mountain site. At both sites, adult emergence started in May. At the mountain site, recruitment started in late July and continued into autumn. There was cohort splitting in the young generation. Some individuals grew rapidly until October-November, but last instar larvae first appeared in March the next year. 1600 degree-days above 0 °C were accumulated during complete development. At the lower site, recruitment began in early July, and cohort splitting also occurred. Fast growing summer recruits emerged as adults in late August, having accumulated only 700 degree-days (above 0 °C). Their offspring hatched in November-December and emerged the next spring, having accumulated also only 700 degree-days. However, only part of the population was bivoltine. Many of the summer recruits grew more slowly and accumulated close to 1900 degree days until they emerged the next spring, together with the offspring of their own fastgrowing siblings. Dependence of growth rate on temperature could not be estimated and appears to vary with daylength. For example, 3-6 °C support growth and development provided daylength exceeds 10 hrs of light, or is rising. At both sites and in all cohorts individuals emerging earliest were larger than later emerging ones. The size decline is significantly correlated with number of days after the winter solstice. For the first time it is shown that the decline does not occur shortly before adult emergence but actually takes place several instars before the last. Size differences are then carried on, and amplified, during subsequent molts, until adulthood. The literature presently relates seasonal size declines of insects to high or rising temperatures experienced by larvae approaching adulthood. Our data show that, at least in Nemurella, this explanation fails. On average, females were distinctly larger than males. Differences in mean last instar size were noticed also between sites and years. They remain presently unexplained. The mean sex ratio in both populations was close to 1:1

The development of Nemurella pictetii KLAPÁLEK (Plecoptera: Nemouridae) in two springstreams in central Europe

From April 1997 to June 1998 Nemurella pictetii populations were regularly sampled in two springstreams at 220 and 850 m a.s.l., respectively, in Hesse (Germany), at approximately 51°N. Random samples of larvae were taken at three week intervals during the vegetation period, and once a month during winter. Sex, instar, body length, head capsule width and wing pad lenght of all larvae were recorded. Temperatures were recorded every hour, temporal patterns of temperature agreed closely between sites. Mean winter lows were 3.9 °C at both sites, the mean summer high was 11.9 °C at the lower site, as opposed to 9.6 °C at the mountain site. At both sites, adult emergence started in May. At the mountain site, recruitment started in late July and continued into autumn. There was cohort splitting in the young generation. Some individuals grew rapidly until October-November, but last instar larvae first appeared in March the next year. 1600 degree-days above 0 °C were accumulated during complete development. At the lower site, recruitment began in early July, and cohort splitting also occurred. Fast growing summer recruits emerged as adults in late August, having accumulated only 700 degree-days (above 0 °C). Their offspring hatched in November-December and emerged the next spring, having accumulated also only 700 degree-days. However, only part of the population was bivoltine. Many of the summer recruits grew more slowly and accumulated close to 1900 degree days until they emerged the next spring, together with the offspring of their own fastgrowing siblings. Dependence of growth rate on temperature could not be estimated and appears to vary with daylength. For example, 3-6 °C support growth and development provided daylength exceeds 10 hrs of light, or is rising. At both sites and in all cohorts individuals emerging earliest were larger than later emerging ones. The size decline is significantly correlated with number of days after the winter solstice. For the first time it is shown that the decline does not occur shortly before adult emergence but actually takes place several instars before the last. Size differences are then carried on, and amplified, during subsequent molts, until adulthood. The literature presently relates seasonal size declines of insects to high or rising temperatures experienced by larvae approaching adulthood. Our data show that, at least in Nemurella, this explanation fails. On average, females were distinctly larger than males. Differences in mean last instar size were noticed also between sites and years. They remain presently unexplained. The mean sex ratio in both populations was close to 1:1