Salinity-induced changes in protein expression in the halophytic plant Nitraria sphaerocarpa

Salinity is a major abiotic stress that inhibits plant growth and development. Plants have evolved complex adaptive mechanisms that respond to salinity stress. However, an understanding of how plants respond to salinity stress is far from being complete. In particular, how plants survive salinity stress via alterations to their intercellular metabolic networks and defense systems is largely unknown. To delineate the responses of Nitraria sphaerocarpa cell suspensions to salinity, changes in their protein expression patterns were characterized by a comparative proteomic approach. Cells that had been treated with 150 mM NaCl for 1, 3, 5, 7, or 9 days developed several stress-related phenotypes, including those affecting morphology and biochemical activities. Of ~1100 proteins detected in 2-DE gel patterns, 130 proteins showed differences in abundance with more than 1.5-fold when cells were stressed by salinity. All but one of these proteins was identified by MS and database searching. The 129 spots contained 111 different proteins, including those involved in signal transduction, cell rescue/defense, cytoskeleton and cell cycle, protein folding and assembly, which were the most significantly affected. Taken together, our results provide a foundation to understand the mechanism of salinity response.     

Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.     

TRIM11 cooperates with HSF1 to suppress the anti-tumor effect of proteotoxic stress drugs

Cells mainly rely on stress proteins, such as heat-shock proteins (HSPs), to respond to various proteotoxic conditions. These proteins protect tumor cells and enhance their survive. However, the regulation of stress proteins involved in protein quality control (PQC) is still poorly understood. Here, we report that the expression of TRIM11, an important regulator of PQC, is positively correlated with tumor cell surviaval during the proteotoxic conditions induced by anti-tumor drugs. In addition, HSF1 is required for TRIM11-mediated removal of protein aggregates and resistance of proteotoxic stress. During these processes, TRIM11 interacts with and stabilizes HSF1, increaseing HSF1 levels in the nucleus. These findings identify that TRIM11, through cooperation with HSF1, protects cells against the proteotoxic stress and promotes tumor cell survival.     

Features and functions of covalently linked proteins in fungal cell walls

The cell walls of many ascomycetous yeasts consist of an internal network of stress-bearing polysaccharides, which serve as a scaffold for a dense external layer of glycoproteins. GPI-modified proteins are the most abundant cell wall proteins and often display a common organization. Their C-terminus can link them covalently to the polysaccharide network, they possess an internal serine- and threonine-rich spacer domain, and the N-terminal region contains a functional domain. Other proteins bind to the polysaccharide network through a mild-alkali-sensitive linkage. Many cell wall proteins are carbohydrate/glycan-modifying enzymes; adhesion proteins are prominent; proteins involved in iron uptake are present, and also specialized proteins that probably help the fungus to survive in its natural environment. The protein composition of the cell wall depends on environmental conditions and developmental stage. We present evidence that the cell wall of mycelial species of the Ascomycotina is similarly organized and contains glycoproteins with comparable functions.     

Protein export from Plasmodium parasites

Many prokaryotic and eukaryotic intracellular pathogens survive by altering the host cell through the export of proteins. In contrast to the well-studied prokaryotic export systems, knowledge of protein export in eukaryotic pathogens is scant. The recent discovery that a short protein sequence targets a protein for export from the malaria parasite Plasmodium falciparum has shed light on the possible mechanism of proteins export and has allowed the preliminary identification of several hundred exported proteins. Among the exported proteins are the members of the paralogous protein families, previously identified exported proteins and many uncharacterized proteins. The interaction of the parasite with the host cell is thus much more complex, and involves more parasite proteins, than previously thought.     

Survival of Animal Tissue Cells in Primary Culture in the Absence of Serum

The ability of cells from tissues of several species of animals to survive in primary culture without serum was tested. Of the species tested, cells from the kidneys of Macaca mulatta (rhesus) and Cercopithecus aethiops (vervet) monkeys and chicken embryo cells not only survived under these conditions, but indeed developed into confluent monolayer cultures. The addition of either serum or its globulin or albumin fraction enhanced the development of cell monolayers and permitted those cells unable to survive in the absence of serum to do so. Certain specific protein trypsin-inhibitors not of serum origin were unable to provide conditions necessary for cell survival or growth when used in place of serum proteins.     

Considerations in predicting phenotypic modifications in amino acid profiles of total cell protein of microorganisms.

A mathematical model is presented that describes the concentration of an amino acid in total cell protein as a function of its concentration in individual cell proteins or in sets of cell proteins. The resulting equation makes it possible to calculate how the makeup of cell proteins must change to obtain a specified alteration in the content of an amino acid in the total cell protein. It is recognized that protein species or sets of proteins that are distinguished by being richer or poorer in a key amino acid than the overall protein must undergo considerable variations in content. The necessary extent of these shifts suggests that the amino acid composition of total cell protein is not likely to be affected significantly by variations in the cultivation conditions.     

A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and cell division

Homologues of CgtA, the common GTP-binding protein of Vibrio harveyi, are present in diverse organisms ranging from bacteria to humans. In bacteria, proteins homologous to CgtA form a subfamily of small GTP-binding proteins, called Obg/Gtp1. Similarity between bacterial members of this subfamily and their eukaryotic homologues is as high as about 50%. Nevertheless, specific functions of these proteins remain largely unknown. Genes coding for CgtA-like proteins are essential in almost all species of bacteria. The only known exception is V. harveyi, whose cells survive disruption of the cgtA gene. Therefore, the V. harveyi cgtA insertional mutant is a very useful tool for studies on functions of CgtA. Here we demonstrate that under normal growth conditions, cells of the cgtA mutant are slightly larger than wild-type cells, whereas indirect inhibition of DNA replication initiation by addition of rifampicin results in significantly higher differences in average cell size between these two strains as measured by flow cytometry. These differences decreased when cell division was inhibited by cephalexin. DNA synthesis per cell mass was found to be increased in the cgtA mutant relative to wild-type V. harveyi strain, whereas the mutant cells grew slower than bacteria with functional cgtA gene. Kinetics of DNA replication after inhibition of cell division was also considerably different in wild-type and cgtA mutant strains. These results suggest that the cgtA gene product plays a role in coupling of DNA replication to cell growth and cell division.     

Proteomic expression profiles of virulent and avirulent strains of Listeria monocytogenes isolated from macrophages

Listeria monocytogenes is able to survive and proliferate within macrophages. In the current study, the ability of three L. monocytogenes strains (serovar 1/2a strain EGDe, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1 was analyzed. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGDe and F2365 proliferated within macrophages for at least 7 h. We further analyzed these strains by comparing their protein expression profiles at 0 h, 3 h, and 5 h post-infection using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. Our results indicated that similar metabolic and cell wall associated proteins were expressed by all three strains at 3 h post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 h post-infection. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive in macrophages.     

Protein regulation mechanism of cold tolerance in Haemaphysalis longicornis

Ticks are external parasitic arthropods that can transmit a variety of pathogens by sucking blood. Low-temperature tolerance is essential for ticks to survive during the cold winter. Exploring the protein regulation mechanism of low-temperature tolerance of Haemaphysalis longicornis could help to explain how ticks survive in winter. In this study, the quantitative proteomics of several tissues of H. longicornis exposed to low temperature were studied by data independent acquisition technology. Totals of 3 699, 3 422, and 1 958 proteins were identified in the salivary gland, midgut, and ovary, respectively. The proteins involved in energy metabolism, cell signal transduction, protein synthesis and repair, and cytoskeleton synthesis changed under low-temperature stress. The comprehensive analysis of the protein regulation of multiple tissues of female ticks exposed to low temperature showed that maintaining cell homeostasis, maintaining cell viability, and enhancing cell tolerance were the most important means for ticks to maintain vital signs under low temperature. The expression of proteins involved in and regulating the above cell activities was the key to the survival of ticks under low temperatures. Through the analysis of a large amount of data, we found that the expression levels of arylamine N-acetyltransferase, inositol polyphosphate multikinase, and dual-specificity phosphatase were up-regulated under low temperature. We speculated that they might have important significance in low-temperature tolerance. Then, we performed RNA interference on the mRNA of these 3 proteins, and the results showed that the ability of female ticks to tolerate low temperatures decreased significantly.     

Biosynthesis, localization, and macromolecular arrangement of the Plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ～600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.     

Glucose Deprivation Induces G2/M Transition-Arrest and Cell Death in N-GlcNAc2-Modified Protein-Producing Renal Carcinoma Cells

Some cancer cells can survive under glucose deprivation within the microenvironment of a tumor. Recently, we reported that N-linked (β-N-acetylglucosamine)₂ [N-GlcNAc₂]-modified proteins induce G2/M arrest and cell death under glucose deprivation. Here, we investigated whether such a response to glucose deprivation contributes to the survival of renal cell carcinomas, which are sensitive to nutritional stress. Specifically, we analyzed seven renal carcinoma cell lines. Four of these cell lines produced N-GlcNAc₂-modified proteins and led G2/M-phase arrest under glucose deprivation, leading to cell death. The remaining three cell lines did not produce N-GlcNAc₂-modified proteins and undergo G1/S-phase arrest under glucose deprivation, leading to survival. The four dead cell lines displayed significant up-regulation in the UDP-GlcNAc biosynthesis pathway as well as increased phosphorylation of p53, which was not observed in the surviving three cell lines. In addition, the four dead cell lines showed prolonged up-regulated expression of ATF3, which is related to unfolded protein response (UPR), while the surviving three cell lines showed only transient up-regulation of ATF3. In this study, we demonstrated that the renal carcinoma cells which accumulate N-GlcNAc₂-modified proteins under glucose deprivation do not survive with abnormaly prolonged UPR pathway. By contrast, renal carcinoma cells that do not accumulate N-GlcNAc₂-modified proteins under these conditions survive. Morover, we demonstrated that buformin, a UPR inhibitor, efficiently reduced cell survival under conditions of glucose deprivation for both sensitive and resistant phenotypes. Further studies to clarify these findings will lead to the development of novel chemotherapeutic treatments for renal cancer.     

Icthyophthirius multifiliis has membrane-associated immobilization antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival

Neurons are post-mitotic cells that undergo an active downregulation of cell cycle-related proteins to survive. The activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells, plays a relevant role in post-mitotic neurons. Recent advances in the study of the regulation of APC/C have documented that the APC/C-activating cofactor, Cdh1, is essential for the function(s) of APC/C in neuronal survival. Here, we review the normal regulation of APC/C activity in proliferating cells and neurons. We conclude that in neurons the APC/C-Cdh1 complex actively downregulates the stability of the cell cycle protein cyclin B1 and the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3. Keeping these proteins destabilized is critical both for preventing the aberrant reentry of post-mitotic neurons into the cell cycle and for maintaining their reduced antioxidant status. Further understanding of the pathophysiological regulation of these proteins by APC/C-Cdh1 in neurons will be important for the search for novel therapeutic targets against neurodegeneration.     

Escaping cell death: survival proteins in cancer

Defects in apoptosis signaling pathways are common in cancer cells. Such defects may play an important role in tumor initiation because apoptosis normally eliminates cells with damaged DNA or dysregulated cell cycle, i.e., cells with increased malignant potential. Moreover, impaired apoptosis may enhance tumor progression and promote metastasis by enabling tumor cells to survive the transit in the bloodstream and to grow in ectopic tissue sites lacking the otherwise required survival factors. Finally, raised apoptosis threshold may have deleterious consequences by rendering cancer cells resistant to various forms of therapy. The intensive apoptosis research during the past decade has resulted in the identification of several proteins which may promote tumorigenesis by inhibiting apoptosis. Of special relevance in human cancer are those commonly expressed in primary tumors and functioning at the common part of the signaling pathway leading to apoptosis. Proteins fulfilling these criteria include antiapoptotic members of the Bcl-2 protein family, heat shock proteins, Hsp70 and Hsp27, as well as survivin, the novel cancer-associated member of the inhibitor of apoptosis protein family. Understanding the molecular mechanisms of action of these proteins may offer novel modes of rationally and selectively manipulating the sensitivity of cancer cells to therapy.     

Ichthyophthirius multifiliis Has Membrane-Associated Immobilization Antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis , immobilize the parasite in vitro. In order to identify ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1 - and 2-dimensiona! SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK

In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane-associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N-terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.     

Modification of the envelope and the protein content of Escherichia coli surviving in sea water

A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy. These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm. These modifications were accompanied by a decrease in cell protein content of approximately 40%. Certain cytoplasmic membrane proteins were lost, and new ones appeared. The development of these changes was considerably slower in cells that had previously been grown in a seawater medium. This suggests that osmotic regulation mechanisms, which enable E. coli to survive much longer in marine conditions, may have a protective influence.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.