Differential protein expression of murine macrophages upon interaction with Candida albicans

Numerous studies highlight the importance of macrophages for optimal host protection against systemic Candida albicans infections. We chose the murine macrophage cell line RAW 264.7 and the wild-type strain C. albicans SC5314 to study of the induced expression/repression of proteins in macrophages when they are in contact with C. albicans, based on 2-DE, comparison between different gels and protein identification. RAW 264.7 cells were allowed to interact with C. albicans cells for 45 min, and a significant differential protein expression was observed in these macrophages compared to controls. Gels were stained with SYPRO Ruby, allowing a better quantification of the intensity of the protein spots. Fifteen spots were up-regulated, whereas 32 were down-regulated; 60 spots appeared and 49 disappeared. Among them, we identified 11 proteins: annexin I, LyGDI (GDID4), Hspa5 (Grp78, Bip), tropomyosin 5 and L-plastin, that augment; and Eif3s5, Hsp60, Hspa9a, Grp58 (ER75), and Hspa8a (Hsc70), that decrease. The translation elongation factor (Eef2p) is modified in some of its different protein species. Many processes seem to be affected: cytoskeletal organisation, oxidative responses (superoxide and nitric oxide production) and protein biosynthesis and refolding.     

Role of CaBud6p in the polarized growth of Candida albicans

Bud6p is a component of a polarisome that controls cell polarity in Saccharomyces cerevisiae. In this study, we investigated the role of the Candida albicans Bud6 protein (CaBud6p) in cell polarity and hyphal development. CaBud6p, which consists of 703 amino acids, had 37% amino-acid sequence identity with the Bud6 protein of S. cerevisiae. The homozygous knock-out of CaBUD6 resulted in several abnormal phenotypes, such as a round and enlarged cells, widened bud necks, and a random budding pattern. In hypha-inducing media, the mutant cells had markedly swollen tips and a reduced ability to switch from yeast to hypha. In addition, a yeast two-hybrid analysis showed a physical interaction between CaBud6p and CaAct1p, which suggests that CaBud6p may be involved in actin cable organization, like Bud6p in S. cerevisiae. Taken together, these results indicate that CaBud6 plays an important role in the polarized growth of C. albicans.     

Alterations of protein expression in macrophages in response to Candida albicans infection

Although macrophages are an important first line of cellular defense, they are unable to effectively kill phagocytosed C. albicans. To determine the physiological basis of this inability, we investigated the alterations of macrophage proteins caused by C. albicans infection. Since the formation of C. albicans hyphae caused cell death, proteins were prepared 3 h after infection and examined by two-dimensional gel electrophoresis (2-DE). The most prominent changes were in glycolytic enzymes, which could have caused energy depletion of the infected cells. Also changed were proteins involved in maintenance of cellular integrity and NO production. Treatment of the macrophages with either cytochalasin D or taxol did not alter their inability to kill C. albicans. Our results indicate that multiple factors contribute to cell death as the pathogenic form of C. albicans becomes fully active inside macrophage cells.     

The role of the unfolded protein response in arrhythmias

Human heart failure is characterized by arrhythmogenic electrical remodeling consisting mostly of ion channel downregulations. Reversing these downregulations is a logical approach to antiarrhythmic therapy, but understanding the pathophysiological mechanisms of the reduced currents is crucial for finding the proper treatments. The unfolded protein response (UPR) is activated by endoplasmic reticulum (ER) stress and has been found to play pivotal roles in different diseases including neurodegenerative diseases, diabetes mellitus, and heart disease. Recently, the UPR is reported to regulate multiple cardiac ion channels, contributing to arrhythmias in heart disease. In this review, we will discuss which UPR modulators and effectors could be involved in regulation of cardiac ion channels in heart disease, and how the understanding of these regulating mechanisms may lead to new antiarrhythmic therapeutics that lack the proarrhythmic risk of current ion channel blocking therapies.     

Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p

We have shown that the unfolded protein response (UPR) in Pichia pastoris requires splicing of a non-conventional intron in the HAC1(u) mRNA in common with other eukaryotes. P. pastoris is a favoured yeast expression host for secreted production of heterologous proteins and the regulation of the UPR in P. pastoris may hold the key to its effective folding and secretion of proteins. We have also shown that the C-terminal region of the Hac1p from P. pastoris is required for functionality. Although the C-terminal regions of Hac1p from both S. cerevisiae and P. pastoris are rich in phenylalanine residues, the P. pastoris Hac1p lacks a C-terminal serine that is known to be important in the efficient functionality of Hac1p from S. cerevisiae.     

Roles of Ras1 membrane localization during Candida albicans hyphal growth and farnesol response

Many Ras GTPases localize to membranes via C-terminal farnesylation and palmitoylation, and localization regulates function. In Candida albicans, a fungal pathogen of humans, Ras1 links environmental cues to morphogenesis. Here, we report the localization and membrane dynamics of Ras1, and we characterize the roles of conserved C-terminal cysteine residues, C287 and C288, which are predicted sites of palmitoylation and farnesylation, respectively. GFP-Ras1 is localized uniformly to plasma membranes in both yeast and hyphae, yet Ras1 plasma membrane mobility was reduced in hyphae compared to that in yeast. Ras1-C288S was mislocalized to the cytoplasm and could not support hyphal development. Ras1-C287S was present primarily on endomembranes, and strains expressing ras1-C287S were delayed or defective in hyphal induction depending on the medium used. Cells bearing constitutively activated Ras1-C287S or Ras1-C288S, due to a G13V substitution, showed increased filamentation, suggesting that lipid modifications are differentially important for Ras1 activation and effector interactions. The C. albicans autoregulatory molecule, farnesol, inhibits Ras1 signaling through adenylate cyclase and bears structural similarities to the farnesyl molecule that modifies Ras1. At lower concentrations of farnesol, hyphal growth was inhibited but Ras1 plasma membrane association was not altered; higher concentrations of farnesol led to mislocalization of Ras1 and another G protein, Rac1. Furthermore, farnesol inhibited hyphal growth mediated by cytosolic Ras1-C288SG13V, suggesting that farnesol does not act through mechanisms that depend on Ras1 farnesylation. Our findings imply that Ras1 is farnesylated and palmitoylated, and that the Ras1 stimulation of adenylate cyclase-dependent phenotypes can occur in the absence of these lipid modifications.     

The role of the unfolded protein response in dengue virus pathogenesis

Symptomatic dengue virus (DENV) infections range from mild fever to severe haemorrhagic disease and death. Host‐viral interactions play a significant role in deciding the fate of the infection. The unfolded protein response (UPR) is a prosurvival cellular reaction induced in response to DENV‐mediated endoplasmic reticulum stress. The UPR has complex interactions with the cellular autophagy machinery, apoptosis, and innate immunity. DENV has evolved to manipulate the UPR to facilitate its replication and to evade host immunity. Our knowledge of this intertwined network of events is continuously developing. A better understanding of the UPR mediated antiviral and proviral effects will shed light on dengue disease pathogenesis and may help development of anti‐DENV therapeutics. This review summarizes the role of the UPR in viral replication, autophagy, and DENV‐induced inflammation to describe how a host response contributes to DENV pathogenesis.     

Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich Syndrome protein homolog Wal1p

The yeast-to-hypha transition is a key feature in the cell biology of the dimorphic human fungal pathogen Candida albicans. Reorganization of the actin cytoskeleton is required for this dimorphic switch in Candida. We show that C. albicans WAL1 mutants with both copies of the Wiskott-Aldrich syndrome protein (WASP) homolog deleted do not form hyphae under all inducing conditions tested. Growth of the wild-type and wal1 mutant strains was monitored by in vivo time-lapse microscopy both during yeast-like growth and under hypha-inducing conditions. Isotropic bud growth produced round wal1 cells and unusual mother cell growth. Defects in the organization of the actin cytoskeleton resulted in the random localization of actin patches. Furthermore, wal1 cells exhibited defects in the endocytosis of the lipophilic dye FM4-64, contained increased numbers of vacuoles compared to the wild type, and showed defects in bud site selection. Under hypha-inducing conditions wal1 cells were able to initiate polarized morphogenesis, which, however, resulted in the formation of pseudohyphal cells. Green fluorescent protein (GFP)-tagged Wal1p showed patch-like localization in emerging daughter cells during the yeast growth phase and at the hyphal tips under hypha-inducing conditions. Wal1p-GFP localization largely overlapped with that of actin. Our results demonstrate that Wal1p is required for the organization of the actin cytoskeleton and hyphal morphogenesis in C. albicans as well as for endocytosis and vacuole morphology.     

Thermotolerance and the heat-shock response in Candida albicans

At elevated temperatures, yeast cells of Candida albicans synthesized nine heat-shock proteins (HSPs) with apparent molecular masses of 98, 85, 81, 76, 72, 54, 34, 26 and 18 kDa. The optimum temperature for the heat-shock response was 45 degrees C although HSPs were detected throughout the range 41-46 degrees C. Protein synthesis was not observed in cells kept at 48 degrees C. Yeast cells survived exposure to an otherwise lethal temperature of 55 degrees C when they had previously been exposed to 45 degrees C. The thermotolerance induced during incubation at 45 degrees C required protein synthesis, since protection was markedly reduced by trichodermin. Mercury ions induced a set of three stress proteins, one of which corresponded in size to an HSP, and cadmium ions evoked one stress protein seemingly unrelated to the HSPs observed after temperature shift.     

MAPK转导通路在白念珠菌菌体抗氧化应激中的作用

目的探讨MAPK通路在念珠菌抗氧化应激中的作用。方法采用不同浓度过氧化氢刺激白念珠菌,通过流式细胞仪检测念珠菌的凋亡率,并计算其增殖指数;通过实时荧光定量PCR检测MAPK通路中8种基因的表达水平。结果随着过氧化氢的刺激浓度增高,白念珠菌的凋亡率逐渐升高,而其增殖指数下降。在不同的过氧化氢浓度刺激下,MAPK通路中各基因表达水平基本一致,即在较低的过氧化氢浓度刺激下,各基因表达水平均有一定的上升,而随着浓度增高,在高浓度的过氧化氢刺激下,各基因表达水平趋于稳定。结论在低浓度的过氧化氢刺激下,白念珠菌的凋亡率虽有所上升,但其相应的增殖指数也有所上升,即生长加快。这可能与其MAPK通路中各基因表达增强有一定的关系。     

The unfolded protein response and its role in intestinal homeostasis and inflammation

The unfolded protein response (UPR) is a signaling pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from the stress caused by misfolded or unfolded proteins [1, 2]. As such, ER stress is an ongoing challenge for all cells given the central biologic importance of secretion as part of normal physiologic functions. This is especially the case for cells that are highly dependent upon secretory function as part of their major duties. Within mucosal tissues, the intestinal epithelium is especially dependent upon an intact UPR for its normal activities [3]. This review will discuss the UPR and the special role that it provides in the functioning of the intestinal epithelium and, when dysfunctional, its implications for understanding mucosal homeostasis and intestinal inflammation, as occurs in inflammatory bowel disease (IBD).