Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

The Viking mission: Implications for life on Mars

Summary The results of the Viking Biology experiments are best explained by non-biological phenomena: The interaction of the reagents with the materials comprising the regolith. Conditions of water activity, temperature, availability of carbon sources and others in most regions of the planet are too extreme for survival and growth of any known Earth microorganisms. Although the possibility persists that some very unusual form of life is somewhere on that planet the evidence is best interpreted as negative. Even though there is no evidence for current life on Mars, whether or not life ever originated there is not known.     

Protein-catalyzed phospholipid exchange in bilayer vesicles determined by flow cytometry and electron microscopy

The protein-induced lipid transfer between phosphatidylcholine vesicles was investigated. Measurements of the degree of polarization at single vesicles were made by flow cytometry using diphenylhexatriene as the optical probe. Vesicles differing in phase transition temperature could be distinguished by their degree of polarization at a temperature where one population was in the fluid ($\text{T > T}{}_{\mathrm{<mtext>t</mtext>}}$) and the other one in the quasi-crystalline ($\text{T < T}{}_{\mathrm{<mtext>t</mtext>}}$) state. Besides vesicles containing exchanged lipids we also observed fractions of unaffected vesicles. The lipid exchange was visualized directly by freeze-fracture electron microscopy. The characteristic ‘ripple’ structure of phosphatidylcholine vesicles disappeared upon exchange with lipid in the fluid state.     

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

The Src Homology 3 Domain-containing Guanine Nucleotide Exchange Factor Is Overexpressed in High-grade Gliomas and Promotes Tumor Necrosis Factor-like Weak Inducer of Apoptosis-Fibroblast Growth Factor-inducible 14-induced Cell Migration and Invasion via Tumor Necrosis Factor Receptor-associated Factor 2

Glioblastoma (GB) is the highest grade of primary adult brain tumors, characterized by a poorly defined and highly invasive cell population. Importantly, these invading cells are attributed with having a decreased sensitivity to radiation and chemotherapy. TNF-like weak inducer of apoptosis (TWEAK)-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival and is dependent upon the activity of multiple Rho GTPases, including Rac1. Here we report that Src homology 3 domain-containing guanine nucleotide exchange factor (SGEF), a RhoG-specific guanine nucleotide exchange factor, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB tumors inversely correlate with patient survival. SGEF mRNA expression is increased in GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo. Furthermore, we showed that, upon TWEAK stimulation, SGEF is recruited to the Fn14 cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of TNF receptor-associated factor 2 (TRAF2) expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14 and inhibited SGEF activity and subsequent GB cell migration. We also showed that knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is an important downstream regulator of TWEAK-Fn14-driven GB cell migration and invasion.     

NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Na⁺/H⁺ exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.