The plasma membrane calcium ATPase MCA-3 is required for clathrin-mediated endocytosis in scavenger cells of Caenorhabditis elegans

Plasma membrane Ca2+ ATPases (PMCAs) maintain proper intracellular Ca2+ levels by extruding Ca2+ from the cytosol. PMCA genes and splice forms are expressed in tissue-specific patterns in vertebrates, suggesting that these isoforms may regulate specific biological processes. However, knockout mutants die as embryos or undergo cell death; thus, it is unclear whether other cell processes utilize PMCAs or whether these pumps are largely committed to the control of toxic levels of calcium. Here, we analyze the role of the PMCA gene, mca-3, in Caenorhabditis elegans. We report that partial loss-of-function mutations disrupt clathrin-mediated endocytosis in a class of scavenger cells called coelomocytes. Moreover, components of early endocytic machinery are mislocalized in mca-3 mutants, including phosphatidylinositol-4,5-bisphosphate, clathrin and the Eps15 homology (EH) domain protein RME-1. This defect in endocytosis in the coelomocytes can be reversed by lowering calcium. Together, these data support a function for PMCAs in the regulation of endocytosis in the C. elegans coelomocytes. In addition, they suggest that endocytosis can be blocked by high calcium levels.     

A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.     

Comparative proteomic network signatures in seminal plasma of infertile men as a function of reactive oxygen species

Background

Reactive oxygen species (ROS) plays a major role in the pathology of male infertility. It is an independent biomarker of sperm function. Seminal plasma is a natural reservoir of antioxidants responsible for the nourishment, protection, capacitation, and motility of sperm within the female reproductive tract resulting in successful fertilization and implantation of the embryo. A comparative proteomic analysis of seminal plasma proteins from fertile men and infertile men with varying levels of ROS was carried out to identify signature proteins involved in ROS-mediated reproductive dysfunction.

Methods

A total of 42 infertile men presenting with infertility and 17 proven fertile donors were enrolled in the study. ROS levels were measured in the seminal ejaculates by chemiluminescence assay. Infertile men were subdivided into Low ROS (0–<93 RLU/s/10⁶ sperm; n = 11), Medium ROS (>93–500 RLU/s/10⁶ sperm; n = 17) and High ROS (>500 RLU/s/10⁶ sperm; n = 14) groups and compared with fertile men (4–50 RLU/s/10⁶ sperm). 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. 1D gel electrophoresis followed by in-gel digestion and LC/MS–MS in a LTQ-Orbitrap Elite hybrid mass spectrometer system was used for proteome analysis. Identification of differentially expressed proteins (DEPs), their cellular localization and involvement in different pathways were examined utilizing bioinformatics tools.

Results

The results indicate that proteins involved in biomolecule metabolism, protein folding and protein degradation are differentially modulated in all three infertile patient groups in comparison to fertile controls. Membrane metallo-endopeptidase (MME) was uniformly overexpressed (>2 fold) in all infertile groups. Pathway involving 35 focus proteins in post-translational modification of proteins, protein folding (heat shock proteins, molecular chaperones) and developmental disorder was overexpressed in the High ROS group compared with fertile control group. MME was one of the key proteins in the pathway. FAM3D was uniquely expressed in fertile group.

Conclusion

We have for the first time demonstrated the presence of 35 DEPs of a single pathway that may lead to impairment of sperm function in men with Low, Medium or High ROS levels by altering protein turn over. MME and FAM3D along with ROS levels in the seminal plasma may serve as good markers for diagnosis of male infertility.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9094-5) contains supplementary material, which is available to authorized users.     

Tempol protects blood proteins and lipids against peroxynitrite-mediated oxidative damage

Oxidative stress is characterized by excessive production of various free radicals and reactive species among which, peroxynitrite is most frequently produced in several pathological conditions. Peroxynitrite is the product of the superoxide anion reaction with nitric oxide, which is reported to take place in the intravascular compartment. Several studies have reported that peroxynitrite targets red blood cells, platelets and plasma proteins, and induces various forms of oxidative damage. This in vitro study was designed to further characterize the types of oxidative damage induced in platelets and plasma proteins by peroxynitrite. This study also determined the ability of tempol to protect blood plasma and platelets against peroxynitrite-induced oxidative damage. The ability of various concentrations of tempol (25, 50, 75, and 100 µM) to antagonize peroxynitrite-induced oxidation was evaluated by measuring the levels of protein carbonyl groups and thiobarbituric-acid-reactive substances in experimental groups. Exposure of platelets and plasma to 100 µM peroxynitrite resulted in an increased levels of carbonyl groups and lipid peroxidation (P < 0.05). Tempol significantly inhibited carbonyl group formation in plasma and platelet proteins (P < 0.05). In addition, tempol significantly reduced the levels of lipid peroxidation in both plasma and platelet samples (P < 0.05). Thus, tempol has antioxidative properties against peroxynitrite-induced oxidative damage in blood plasma and platelets.     

Exendin-4类似物的克隆、表达与体内生物活性检测

为研究Exendin-4类似物的克隆,融合表达及在体内的生物活性,在pED载体融合伴侣序列中插入利于下游分离纯化的序列成为5#载体,将Exendin-4类似物基因与5#载体中的融合伴侣基因通过酸水解位点连接,转化至E.coli BL21中并诱导表达融合蛋白,酸水解将目的肽与融合伴侣分开后,经阴离子交换树脂分离得到目的肽。6周～8周正常雄性ICR小鼠皮下注射Exendin-4类似物后,口服糖耐量实验检测在不同时间段小鼠血液中葡萄糖及胰岛素含量的变化。结果表明:融合蛋白的表达量占菌体总蛋白的40%,Exendin-4类似物纯度达91.8%。Exendin-4类似物的活性与对照组相比,具有显著的降低血糖和显著促进胰岛素分泌的活性(P<0.01)。     

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180 min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180 min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.     

CHO cells expressing the human P₅-ATPase ATP13A2 are more sensitive to the toxic effects of herbicide paraquat

P-ATPases are membrane transporters energized by ATP. The subfamily of P₅-ATPases is the least studied P-ATPases and the ion substrate specificity of the P₅ subfamily is not known. Mutations of the human P₅ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.     

Calcium efflux of plasma membrane vesicles exposed to ELF magnetic fields-test of a nuclear magnetic resonance interaction model

The question whether very weak, low frequency magnetic fields can affect biological matter is still under debate. The theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely weak 60 Hz magnetic fields on the transport of Ca²⁺ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested a newly proposed quantum mechanical model postulates that polarization of hydrogen nuclei can elicit a biological effect. Vesicles were exposed for half an hour at 32 °C and the calcium efflux was studied using radioactive ⁴⁵Ca²⁺ as a tracer. A static magnetic field of 26 µT and time‐varying magnetic fields with a frequency of 60 Hz and amplitudes between 0.6 and 6.3 µT were used. The predictions of the model, proposed by Lednev, that at a frequency of 60 Hz the biological effect under investigation would significantly be altered at the amplitudes of 1.3 and 3.9 µT could not be confirmed. Bioelectromagnetics 33:535–542, 2012. © 2012 Wiley Periodicals, Inc.