Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket

The α2 isoform of Na,K-ATPase plays a crucial role in Ca²⁺ handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, “affinity” for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order α2 ≫ α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.     

Digoxin Derivatives with Enhanced Selectivity for the α2 Isoform of Na,K-ATPase: EFFECTS ON INTRAOCULAR PRESSURE IN RABBITS*

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1β1, α2β1, and α3β1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH₂ substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.     

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.     

The proto-oncogene Bcl-2 inhibits cellular toxicity of dopamine: possible implications for Parkinson's Disease

It is currently believed that excessive oxidant stress induced by metabolism of dopamine (DA), plays a major role in the pathogenesis of the selective nigrostriatal neuronal loss in Parkinson's disease. We recently showed that the neurotransmitter DA, in physiological concentrations, is capable of initiating apoptosis in cultured, post-mitotic sympathetic neurons. Bcl-2 is a proto-oncogene that blocks apoptosis. We now report that Bcl-2 is a powerful inhibitor of DA toxicity in PC-12 pheochromocytoma cells. We induced stable expression of Bcl-2 in PC-12 cells by transfection with recombinant pCMV5 expression vector, containing mouse bcl-2 (coding-sequence) cDNA. Cells expressing Bcl-2 manifested marked resistance to otherwise lethal (300 uM) in vitroconcentrations of DA. This protective effect was reflected in the trypan-blue test of cell survival, 3 H-thymidine incorporation and inhibition of the characteristic apoptotic morphologic alterations in scanning electron microscopic studies. Bcl-2 and associated control systems of apoptosis may have an important physiological role in restraining the apop-tosis-triggering potential of DA in nigrostriatal neurons. This novel field of research may yield insights into the pathogenesis of Parkinson's disease and lead to development of novel therapeutic approaches.     

The enzymatic activity of lysyl oxidas-like-2 (LOXL2) is not required for LOXL2-induced inhibition of keratinocyte differentiation

Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis. It also inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. Stimulation of HaCaT skin keratinocytes with exogenous LOXL2 or overexpression of LOXL2 in these cells inhibits their differentiation as manifested by inhibition of calcium or vitamin D-induced involucrin expression. The inhibition was abrogated by the LOXL2 function-blocking monoclonal antibody AB0023 as well as by an anti-LOXL2 polyclonal antibody. Surprisingly, a point-mutated form of LOXL2 (LOXL2(Y689F)) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, also inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity. This conclusion was supported by experiments that showed that β-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. The activity of LOXL2(Y689F) required the presence of the fourth scavenger receptor-cysteine-rich (SRCR) domain of LOXL2, which is also the binding target of AB0023. Epitope-tagged LOXL2(Y689F) was internalized at 37 °C by HaCaT cells. The internalization was inhibited by AB0023 and by competition with unlabeled LOXL2, suggesting that these cells may express a LOXL2 receptor. Our results suggest that agents that inhibit the enzymatic activity of LOXL2 may not suffice to inhibit completely the effects of LOXL2 on complex processes that involve altered states of cellular differentiation.     

Lead concentrates in ovarian follicle compromises pregnancy

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction.     

The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.     

A galling aphid furnishes its home with a built-in pipeline to the host food supply

Galling insects modify the developmental pathways of their host plants and create a protected and favourable microhabitat for their offspring. Galling aphids reproduce in their galls and the resulting clones often comprise hundreds of individuals. We followed the histological changes in the host Pistacia palaestina (Anacardiaceae) induced by the galling aphid Geoica wertheimae (Aphidoidea, Pemphigidae: Fordinae). We showed that the leaf tissues are altered in the gall in a way that gives the aphids easy access to the vascular system from which they obtain their nourishment. Specifically, the cuticle that lines the normal leaf epidermal cells is absent on the surface of the inner gall cavity, and the normal palisade cells are replaced by parenchymatous tissue with numerous wide latex ducts. Associated with these ducts, many new sieve tubes (phloem elements) are formed just a few cell layers below the inner gall surface. This arrangement enables the hundreds of aphids to feed simultaneously and continuously in the gall throughout the summer.