Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Analysis of ZAPs,Zymogen Granule Membrane Associated Proteins,in the Regulated Exocytosis of the Pancreas

To characterize molecules involved in the intracellular sorting and regulated exocytosis of digestive enzymes in the pancreas, proteins that are specifícially associated with the zymogen granule membranes were analyzed. Zymogen granules, the major secretory organelles in the pancreas, were highly purified. SDS-PAGE analysis found at least 7 protein components in the zymogen granule membranes including ZAP (zymogen granule membrane associated protein) 75, 54, 47, 36, 32, 29, 25 (numbers refer to their apparent kDas). ZAP75 is identical to the glycophosphatidylinositol (GPI)-anchored protein, GP2. Partial amino acid sequencing of ZAP47 and ZAP36 found similarities to a preprocarboxypeptidase B and annexins, respectively. The method we used was a useful tool for structural analysis of the members of ZAPs.     

杜氏盐藻(Dunalielle salina)盐适应相关蛋白

观察不同盐浓度培养液中生长的杜氏盐藻可溶性蛋白SDS-PAG图谱,发现高盐与低盐相比,其51kD和63kD蛋白含量高,而23kD的蛋白含量则低。高渗骤变后,51kD蛋白的含量减少,而23kD蛋白的含量增加两倍或两倍以上,骤变23h后含量增加已非常明显;低渗骤变后24h,51kD和23kD蛋白的含量变化不明显。另外,有一分子量约为26kD的蛋白在高盐下易降解。分析了这些蛋白与社氏盐藻渗透调节的可能关系。     

Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.     

Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.     

Trauma of the Erythrocyte Membrane Associated with Low Shear Stress

Studies have been performed on erythrocytes that have been subjected to a low shear stress of less than 100 dyn/cm² in a cone-and-plate viscometer. Alterations that were observed included decreased red cell survival, increased osmotic fragility, changes in the cation permeability of the red cell membrane, and a reduction in membrane-associated acetylcholinesterase activity. Some of these alterations are similar to those accompanying aging. The observed data suggest that one segment of the erythrocyte population is more susceptible to shear-induced damage than the rest of the cells.     

Restoration of Proper Trafficking to the Cell Surface for Membrane Proteins Harboring Cysteine Mutations

A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine’s sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids’ side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems.     

Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti–endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.Polarity is a fundamental characteristic of most eukaryotic cells, either as a transient phenomenon (e.g., in a moving fibroblast) or a permanent feature (e.g., of an epithelial layer) (Drubin and Nelson, 1996). In epithelial cells, polarity is evident at many levels. At the cell surface, the basolateral and apical membrane domains face different environments (internal and external, respectively) and each membrane contains a distinct set of proteins and lipids (Simons and Fuller, 1985). Acquisition of the fully polarized epithelial phenotype requires assembly of tight and adhering junctions, which serve as barriers separating the apical and basolateral surfaces, and the selective delivery of plasma membrane (PM)¹ molecules and/or their retention at each surface (Rodriguez-Boulan and Powell, 1992; Simons et al., 1992; Wollner and Nelson, 1992).There is great variety among epithelial cells in the way specific PM proteins reach the same or different destinations. For example, kidney-derived MDCK cells sort most apical and basolateral membrane components in the TGN and then export this cargo directly to the “correct” surface (Matter and Mellman, 1994), although a variant line was recently found that delivers Na⁺,K⁺-ATPase to all PM domains randomly and then achieves a predominant basolateral distribution by selective retention (Hammerton et al., 1991; Mays et al., 1995). In other epithelial cells, apical PM proteins are first transported to the basolateral surface and then subsequently transcytosed to the apical domain, with sorting occurring in the endocytic pathway. The extent to which this more circuitous or “indirect” pathway to the apical surface is used depends on the specific protein and cell type (Rodriguez-Boulan and Zurzolo, 1993; Matter and Mellman, 1994). For delivery of apical membrane proteins, hepatocytes in vivo appear to use the indirect pathway exclusively (Bartles et al., 1987; Schell et al., 1992; Maurice et al., 1994), whereas cultured HepG2 cells reportedly deliver selected membrane lipids directly from the TGN to the apical PM (Zaal et al., 1994).The structural information directing membrane proteins through the transcytotic pathway has been elucidated only for the polymeric IgA receptor (pIgA-R). It is a sacrificial receptor whose 103-amino acid cytoplasmic tail contains multiple signals that direct the protein through the secretory pathway and into the transcytotic branch of the endocytic system. pIgA-R''s final destination is the apical membrane where an 80-kD proteolytic fragment of the receptor''s ectodomain is released into the apical milieu. An important difference between the pIgA-R and resident apical PM proteins studied so far is that the latter usually have short cytoplasmic tails with no apparent sorting signal (e.g., aminopeptiase N [APN] and dipeptidyl peptidase IV [DPPIV]), or are glycosyl phosphatidyl inositol (GPI)- anchored (e.g., 5′-nucleotidase [5′NT]). Positive sorting information is present elsewhere in these proteins, e.g., the glycolipid anchor of GPI-proteins (Lisanti and Rodriguez-Boulan, 1990) and the large ectodomains of APN and DPPIV (Vogel et al., 1992, 1995; Weisz et al., 1992), but finer resolution of such global signals has not yet been attained.Many studies have described the membrane compartments involved in the basolateral-to-apical transcytosis of soluble and/or membrane-bound cargo (e.g., Bomsel et al., 1989; Brändli et al., 1990; Hayakawa et al., 1990; van Deurs et al., 1990; van Genderen and van Meer, 1995). Although it is now clear that multiple compartments participate, the existence of stations or carriers that are unique to the transcytotic pathway is still an open question (e.g., Barroso and Sztul, 1994, versus Apodaca et al., 1994), as are the number and location(s) of the sorting site(s) for transcytotic cargo versus cargo destined for the recycling or lysosomal branches of the endocytic system (for reviews see Courtoy, 1993; Sandoval and Bakke, 1994; Gruenberg and Maxfield, 1995; Mostov and Cardone, 1995). The remarkable plasticity of the endocytic system as well as the possibility of real differences in the transport of soluble and membrane cargo may explain some of the apparent paradoxes. Early immuno-EM studies reported that in hepatocytes in vivo, the pIgA-R shares clathrin-coated entry sites with receptors that recycle between the PM and endosomal compartments (asialoglycoprotein receptor [ASGP-R] and mannose-6-phosphate receptor [M6P-R]), but is then segregated from them at the level of peripheral endosomes (called compartment for uncoupling of receptors and ligands) (Geuze et al., 1984). In contrast, the entry site(s) for resident apical proteins transiently present at the basolateral surface is still unknown. However, in liver in situ, newly synthesized DPPIV colocalizes with transcytosing pIgA-R in subapical tubulovesicular structures, suggesting that, at least in these cells, the last steps of transcytosis are common (Barr and Hubbard, 1993). Moreover, transcytotic membranes can be isolated that contain pIgA-R and newly synthesized DPPIV (Barr et al., 1995). Nevertheless, the extent to which different membrane protein classes with a common destination share a common pathway is still unclear.The newly developed WIF-B cell line is an ideal in vitro model for studying PM protein trafficking in polarized hepatocytes (Ihrke et al., 1993; Shanks et al., 1994). WIF-B cells grow in monolayers and acquire a polarized phenotype reminiscent of hepatocytes in vivo; that is, neighboring cells form bile canalicular-like spaces (BC). Each BC is completely sequestered from the surrounding medium as well as the substratum and apical PM proteins are highly concentrated in the BC membrane. Tight junctions prevent mixing of apical and basolateral PM proteins and block diffusion of large molecules such as antibodies from the culture medium into the BC (Ihrke et al., 1993).As a first step toward understanding the transcytotic pathway(s) in WIF-B cells and ultimately in liver, we define here the intracellular trafficking pathways taken by three different classes of membrane proteins that pass through the basolateral membrane: (a) apical PM proteins and pIgA-R; (b) basolaterally recycling receptors; and (c) proteins of the endosomal/lysosomal pathway that cycle through the PM. These proteins were tracked in living WIF-B cells by labeling with specific antibodies at the basolateral surface and determining the distributions of the antigen–antibody complexes at later times. Antibodies to a variety of apical PM proteins and the pIgA-R were specifically and efficiently transcytosed from the basolateral to the apical domain; all passed through a prominent subapical compartment before fusion with the apical PM. In contrast, antibodies to cycling membrane proteins, such as the ASGP-R, transferrin receptor (Tf-R), and M6P-R, and the lysosomal membrane protein lgp120, did not appear to pass through the subapical compartment, but rather were directly transported to the intracellular compartments that contained the highest concentrations of their antigens at steady state. However, antibodies to endolyn-78, another endosomal/lysosomal membrane protein (Croze et al., 1989), appeared transiently in the apical region of the cells before accumulating in lysosomes. Thus, the trafficking of endolyn-78 resembled to some degree the transcytotic route of apical PM proteins and pIgA-R.Our observations verify that transcytosis is a pathway for the delivery of apical PM proteins to the apical surface in WIF-B cells, as is seen in hepatocytes in vivo. Our findings suggest that two successive sorting compartments operate in WIF-B cells. Basolaterally endocytosed proteins pass first through peripheral endosomes, the compartment from which most ASGP-R and transferrin receptor (Tf-R) molecules recycle; from there lysosomal proteins such as lgp120 are directed towards lysosomes whereas transcytotic molecules are sorted out for transport to the apical pole. However, segregation of apical residents from at least one endosomal/lysosomal marker, endolyn-78, appears to occur after these proteins are delivered to an endomembrane compartment in the subapical region.²     

Characterization of Outer Membrane Proteins of Escherichia Coli in Response to Phenol Stress

Gram-negative bacteria are generally more tolerant to disinfectants than Gram-positive bacteria due to outer membrane (OM) barrier, but the tolerant mechanism is not well characterized. We have utilized comparative proteomic methodologies to characterize the OM proteins of E. coli K-12 K99+ in response to phenol stress and found that nine proteins were altered significantly. They were OM proteins OmpA, FadL, LamB, and OmpT, cytoplasmic-associated proteins AceA and EF-Tu, inner membrane protein AtpB, putative capsid protein Q8FewO, and unknown location protein Dps. They were reported here for the first time to be phenol-tolerant proteins. The alteration and functional characterization of the four OM proteins were further investigated using western blotting, genetically modified strains with gene deletion and gene complementation approaches. Our results characterized the functional OM proteins of E. coli in resistance to phenol, and provide novel insights into the mechanisms of bacterial disinfectant-tolerance and new drug targets for control of phenol-resistant bacteria.     

Novel Mutations of ABCB6 Associated with Autosomal Dominant Dyschromatosis Universalis Hereditaria

Objective

Dyschromatosis universalis hereditaria (DUH) is a rare heterogeneous pigmentary genodermatosis, which was first described in 1933. The genetic cause has recently been discovered by the discovery of mutations in ABCB6. Here we investigated a Chinese family with typical features of autosomal dominant DUH and 3 unrelated patients with sporadic DUH.

Methods

Skin tissues were obtained from the proband, of this family and the 3 sporadic patients. Histopathological examination and immunohistochemical analysis of ABCB6 were performed. Peripheral blood DNA samples were obtained from 21 affected, 14 unaffected, 11 spouses in the family and the 3 sporadic patients. A genome-wide linkage scan for the family was carried out to localize the causative gene. Exome sequencing was performed from 3 affected and 1 unaffected in the family. Sanger sequencing of ABCB6 was further used to identify the causative gene for all samples obtained from available family members, the 3 sporadic patients and a panel of 455 ethnically-matched normal Chinese individuals.

Results

Histopathological analysis showed melanocytes in normal control’s skin tissue and the hyperpigmented area contained more melanized, mature melanosomes than those within the hypopigmented areas. Empty immature melanosomes were found in the hypopigmented melanocytes. Parametric multipoint linkage analysis produced a HLOD score of 4.68, with markers on chromosome 2q35-q37.2. A missense mutation (c.1663 C>A, p.Gln555Lys) in ABCB6 was identified in this family by exome and Sanger sequencing. The mutation perfectly cosegregated with the skin phenotype. An additional mutation (g.776 delC, c.459 delC) in ABCB6 was found in an unrelated sporadic patient. No mutation in ABCB6 was discovered in the other two sporadic patients. Neither of the two mutations was present in the 455 controls. Melanocytes showed positive immunoreactivity to ABCB6.

Conclusion

Our data add new variants to the repertoire of ABCB6 mutations with DUH.     

Novel Compound Heterozygous Mutations in MYO7A Associated with Usher Syndrome 1 in a Chinese Family

Usher syndrome is an autosomal recessive disease characterized by sensorineural hearing loss, age-dependent retinitis pigmentosa (RP), and occasionally vestibular dysfunction. The most severe form is Usher syndrome type 1 (USH1). Mutations in the MYO7A gene are responsible for USH1 and account for 29–55% of USH1 cases. Here, we characterized a Chinese family (no. 7162) with USH1. Combining the targeted capture of 131 known deafness genes, next-generation sequencing, and bioinformatic analysis, we identified two deleterious compound heterozygous mutations in the MYO7A gene: a reported missense mutation c.73G>A (p.G25R) and a novel nonsense mutation c.462C>A (p.C154X). The two compound variants are absent in 219 ethnicity-matched controls, co-segregates with the USH clinical phenotypes, including hearing loss, vestibular dysfunction, and age-dependent penetrance of progressive RP, in family 7162. Therefore, we concluded that the USH1 in this family was caused by compound heterozygous mutations in MYO7A.     

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.     

Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps

ACA8 is a type 2B Ca²⁺-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu²⁵² to Asn³⁴⁵ sequence. Mutation to Ala of any of six tested acidic residues (Glu²⁵², Asp²⁷³, Asp²⁹¹, Asp³⁰³, Glu³⁰², or Asp³³²) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca²⁺-ATPase plays a role in the attainment of the auto-inhibited state.     

Insulin-induced Glut4 recruitment in the fatty Zucker rat heart is not associated with changes in Glut4 content in the intracellular membrane

Molecular and Cellular Biochemistry - Impaired cardiac glucose metabolism and glucose transport have been shown in the insulin resistant fatty Zucker rat. The aim of the present study was to...