The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.     

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.     

Motherhood and the hormones of pregnancy modify concentrations of hippocampal neuronal dendritic spines

Short-term fluctuations in steroid hormones such as estradiol (E2) and progesterone (P) can affect the concentration of hippocampal dendritic spines in adult, cycling nulliparous female rats. Pregnancy is characterized by a significantly longer duration of substantially elevated E2 and P compared to the estrous cycle. Thus, even greater changes than those reported during estrus may be evident. In two experiments, we examined the extent to which reproductive and hormonal state altered the concentration of apical neuronal dendritic spines of the CA1 region of the hippocampus in the following age-matched groups (N's = 7-10/group) of rats: in Exp. 1., CA1 dendritic spine density was examined in nulliparous diestrus (DES), proestrus (PRO), and estrus (ES) females, and late-pregnant (LP) (day 21) and lactating (day 5-6; LACT) females. In Exp. 2, the effects on spine density of a regimen mimicking pregnancy (and that stimulates maternal behavior) were examined, using ovariectomized, no hormone-exposed (OVX-minus) vs. sequential P&E(2)-treated (OVX + P&E2) groups. For both experiments, brains were removed, Golgi-Cox-stained and the most lateral tertiary branches of the apical dendrite of completely-stained hippocampal CA1 pyramidal neurons were traced with oil-immersion at x 1600 and dendritic spine density (# spines/10 micro dendritic segment) recorded. In Exp. 1, spine density was increased in LP and LACT females (which were not different) compared to the other virgin groups, including PRO females, who had more spines than DES and ES. In Exp. 2, OVX + P&E2 displayed significantly more dendritic spines per 10 micro than OVX-minus females (and had numbers that were similar to those of LP and LACT from Exp. 1). Pregnancy and its attendant hormonal fluctuations, therefore, may alter hippocampal neurons that regulate some non-pup-directed components of maternal behavior (e.g., nest building) or behaviors that support maternal behavior (e.g., foraging, associative memory).     

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.     

The transcriptional profile of the kidney in Tsc2 heterozygous mutant Long Evans (Eker) rats compared to wild-type

Hereditary renal cell carcinoma (RCC) in Eker rats results from an inherited insertional mutation in the Tsc2 tumor suppressor gene and provides a valuable experimental model to characterize the function of the Tsc2 gene product, tuberin in vivo. The Tsc2 mutation predisposes the Eker rat to develop renal tumors at an early age. The exact mechanism of Tsc2 mediated tumor suppression is not known, however, there is evidence that it is most likely mediated by changes in cell cycle regulation via the PI3K/Akt pathway. The present study was designed to identify if gene expression was different in Tsc2 heterozygous mutant rat kidney compared to wild-type and if any of those differences are associated with tumorigenesis. cDNA microarray analysis of the untreated Tsc2 (+/-) mutant Long Evans (Eker) rat was compared to the Tsc2 (+/+) wild-type Long Evans rat to search for patterns that might be indicative of the intrinsic role of Tsc2. Of 4395 genes queried, 3.2% were significantly altered in kidneys from heterozygous mutant rats, of which 110 (76%) were up-regulated and 34 (24%) were down-regulated relative to the wild-type. The genes with altered expression belonged to the functional categories of cell cycle regulation, cell proliferation, cell adhesion and endocytosis. Many of these genes appear to be directly or indirectly regulated by the PI3K/Akt pathway. In addition to the PI3K/Akt pathway, other signaling pathways were also differentially expressed in Tsc2 mutant Eker rat kidneys compared to wild-type rats. The gene expression profiles of the Tsc2 heterozygous mutant and wild-type animals highlights new pathways for investigation that may be associated with the tumorigenic activity of tuberin loss and correlate with the enhanced susceptibility of the Tsc2 mutant animal's tendency to develop renal cell carcinoma.     

Lucky rugose corals on crinoid stems: unusual examples of subepidermal epizoans from the Devonian of Morocco

Berkowski, B & Klug, C. 2011: Lucky rugose corals on crinoid stems: unusual examples of subepidermal epizoans from the Devonian of Morocco. Lethaia, Vol. 45, pp. 24–33. In the fossil record, evidence for true epizoans, i.e. living animals inhabiting other living host‐animals, is rather rare. A host reaction is usually needed to proof the syn vivo‐settling of the epizoan. Herein, we provide a first report of such an epizoan biocoenosis from various strata of the Early Devonian of Hamar Laghdad, the world‐renowned Moroccan mud‐mound locality. In this case, solitary rugose corals settled as larvae on crinoid stems, perhaps at a spot where the epidermis was missing for some reason (injury, disease). Both the crinoid and the coral began to grow around each other. By doing so, the affected crinoid columnals formed a swelling, where ultimately only an opening slightly larger than the coral orifice remained. We discuss both macroecological and small‐scale synecological aspects of this biocoenosis. The coral profited from its elevated home because it reached into more rapid currents providing the polyp with more food than at the densely populated seafloor, which was probably covered by a coral‐meadow around the mounds and hydrothermal vents. □Corals, crinoids, Early Devonian, epizoans, Morocco, Rugosa.