Jennifer Lowe

A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine.

Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport. Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease. Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport. Wild-type Ccc2p and variants were expressed in a ccc2-Δ yeast strain to check whether they were able to restore copper transport by complementation. Wild-type Ccc2p and variants were also expressed in Sf9 cells using baculovirus to study their enzymatic properties on membrane preparations. The latter system allowed us to measure a copper-activated ATPase activity of about 20 nmol/mg/min for the wild-type Ccc2p at 37 °C. None of the variants was as efficient as the wild type in restoring copper homeostasis. The mutation of each cysteine of the 583CPC585 motif into a serine resulted in nonfunctional proteins that could not restore copper homeostasis in yeast and had no ATPase activity. Phosphorylation by ATP was still possible with the C583S variant, although it was not possible with the C585S variant, suggesting that the cysteines of the CPC motif have a different role in copper transport. Cys583 would be necessary for copper dissociation and/or enzyme dephosphorylation and Cys585 would be necessary for ATP phosphorylation, suggesting a role in copper binding.

Renin-angiotensin system in the kidney: What is new?

The renin-angiotensin system (RAS) has been known for more than a century as a cascade that regulates body fluid balance and blood pressure. Angiotensin II(Ang II) has many functions in different tissues; however it is on the kidney that this peptide exerts its main functions. New enzymes, alternative routes for Ang IIformation or even active Ang II-derived peptides have now been described acting on Ang II AT1 or AT2 receptors, or in receptors which have recently been cloned, such as Mas and AT4. Another interesting observation was that old members of the RAS, such as angiotensin converting enzyme (ACE), renin and prorenin, well known by its enzymatic activity, can also activate intracellular signaling pathways, acting as an outside-in signal transduction molecule or on the renin/(Pro)renin receptor. Moreover, the endocrine RAS, now is also known to have paracrine, autocrine and intracrine action on different tissues, expressing necessary components for local Ang II formation. This in situ formation, especially in the kidney, increases Ang II levels to regulate blood pressure and renal functions. These discoveries, such as the ACE2/Ang-(1-7)/Mas axis and its antangonistic effect rather than classical deleterious Ang II effects, improves the development of new drugs for treating hypertension and cardiovascular diseases.

Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase‐mediated phosphorylation. However, there is no evidence showing the influence of kinase‐related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP‐dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)‐ATPase, with PKA‐mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long‐range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high‐energy to the low‐energy phosphorylated intermediate associated with copper release.

Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

Copper delivery to Ccc2 – the Golgi Cu+‐ATPase – was investigated in vivo, replacing the Cu+‐chaperone Atx1 by various structural homologues in an atx1‐Δ yeast strain. Various proteins, displaying the same ferredoxin‐like fold and (M/L)(T/S)CXXC metal‐binding motif as Atx1 and known as Cu+‐, Cd2+‐ or Hg2+‐binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1‐homologues is discussed.

Bone marrow mononuclear cells shift bioactive lipid pattern in injured kidney towards tissue repair in rats with unilateral ureteral obstruction

BACKGROUND Bioactive lipids are important in tissue injury and regeneration. Ceramide (Cer) is known for its pro-apoptotic action and sphingosine-1-phosphate (S1P) for inducing proliferation and cell survival; diacylglycerol (DAG) and lysophosphatidic acid (LPA) are involved in various signalling pathways including modulation of ion transport. LPA signalling through its receptor LPA(1) is also related to the progression of fibrosis. This study investigated the modulation of lipid signalling pathways induced by administration of bone marrow-derived mononuclear cells (BMMC) in chronic kidney disease. METHODS Unilateral ureteral obstruction (UUO) was followed by intravenous injection of ∼2 × 10(7) BMMC. Controls were UUO group treated with buffered solution and sham-operated group. Animals were killed 14 days after surgery, and lipid phosphorylation assays and immunoblotting were performed on the kidney homogenates. RESULTS More DAG was available in the UUO rats (2.4 ± 0.4 and 2.4 ± 0.3 vs 1.0 ± 0.2 pmol (32)PA mg(-)(1) min(-)(1), in UUO and UUO + BMMC vs SHAM). Sphingosine kinase was 150 ± 12% more active in UUO + BMMC than in UUO and SHAM. Cer levels were 76 ± 7% lower in the UUO + BMMC than UUO. LPA receptor type 1 (LPA(1)) expression was 169 ± 7% higher in the UUO group than in UUO + BMMC and SHAM. BMMC maintain control levels of Ca(2+)-ATPase expression altered by UUO by 40%. CONCLUSIONS BMMC infusion modulated diverse lipid signalling pathways and protein expression, shifted sphingolipid metabolism toward a regenerative pattern and favourably reduced the levels of a receptor involved in the progression of tissue fibrosis. These results strengthen the benefits of BMMC treatment and give insight into its paracrine mechanisms of action.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.

Golgi membranes from liver express an ATPase with femtomolar copper affinity, inhibited by cAMP-dependent protein kinase

Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (∼1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.

Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells

ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of β-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of β-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.

Two Serine Residues Control Sequential Steps during Catalysis of the Yeast Copper ATPase through Different Mechanisms That Involve Kinase-mediated Phosphorylations

Ccc2, the yeast copper-transporting ATPase, pumps copper from the cytosol to the Golgi lumen. During its catalytic cycle, Ccc2 undergoes auto-phosphorylation on Asp627 and uses the energy gained to transport copper across the cell membrane. We previously demonstrated that cAMP-dependent protein kinase (PKA) controls the energy interconversion CuE∼P → E-P + Cu when Ser258 is phosphorylated. We now demonstrate that Ser258 is essential in vivo for copper homeostasis in extremely low copper and iron concentrations. The S258A mutation abrogates all PKA-mediated phosphorylations of Ccc2, whereas the S971A mutation leads to a 100% increase in its global regulatory phosphorylation. With S258A, the first-order rate constant of catalytic phosphorylation by ATP decreases from 0.057 to 0.030 s−1, with an 8-fold decrease in the burst of initial phosphorylation. With the S971A mutant, the rate constant decreases to 0.007 s−1. PKAi5–24 decreases the amount of the aspartylphosphate intermediate (EP) in Ccc2 wt by 50% within 1 min, but not in S258A, S971A, or S258A/S971A. The increase of the initial burst and the extremely slow phosphorylation when the “phosphomimetic” mutant S258D was assayed (k = 0.0036 s−1), indicate that electrostatic and conformational (non-electrostatic) mechanisms are involved in the regulatory role of Ser258. Accumulation of an ADP-insensitive form in S971A demonstrates that Ser971 is required to accelerate the hydrolysis of the E-P form during turnover. We propose that Ser258 and Ser971 are under long-range intramolecular, reciprocal and concerted control, in a sequential process that is crucial for catalysis and copper transport in the yeast copper ATPase.

Dissecting copper homeostasis in diabetes mellitus

Diabetes Mellitus (DM) is characterized by elevated blood glucose levels (hyperglycemia). It can occur due to impaired secretion or action of the hormone insulin, which is produced by pancreatic beta‐cells to promote the entry of glucose into the cells. It is known that hyperglycemia has an important role in the production of reactive oxygen species in all types of DM and that an imbalance of transition metal as Cu and Fe plays a pivotal role in stimulating the oxidative stress. Different levels of some transition metals, as Cu, Fe, Mn, and Zn has been reported comparing diabetic animal models with the control group. An increased Cu status is also described in diabetic patients. Homeostasis of Cu depends on distinct proteins, where Cu(I)‐ATPases are important transmembrane proteins for acquisition, active transport, distribution and elimination of Cu ions. In this review we first provide an overview of the literature about the relationship between diabetes and copper, the modulation of Cu(I)‐ATPases activity and protein expression in DM, to next discuss the alternative treatments for diabetes using Cu chelation.