Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response.     

Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.     

Sho1 and Msb2-Related Proteins Regulate Appressorium Development in the Smut Fungus Ustilago maydis

The dimorphic fungus Ustilago maydis switches from budding to hyphal growth on the plant surface. In response to hydrophobicity and hydroxy fatty acids, U. maydis develops infection structures called appressoria. Here, we report that, unlike in Saccharomyces cerevisiae and other fungi where Sho1 (synthetic high osmolarity sensitive) and Msb2 (multicopy suppressor of a budding defect) regulate stress responses and pseudohyphal growth, Sho1 and Msb2-like proteins play a key role during appressorium differentiation in U. maydis. Sho1 was identified through a two-hybrid screen as an interaction partner of the mitogen-activated protein (MAP) kinase Kpp6. Epistasis analysis revealed that sho1 and msb2 act upstream of the MAP kinases kpp2 and kpp6. Furthermore, Sho1 was shown to destabilize Kpp6 through direct interaction with the unique N-terminal domain in Kpp6, indicating a role of Sho1 in fine-tuning Kpp6 activity. Morphological differentiation in response to a hydrophobic surface was strongly attenuated in sho1 msb2 mutants, while hydroxy fatty acid–induced differentiation was unaffected. These data suggest that Sho1 and the transmembrane mucin Msb2 are involved in plant surface sensing in U. maydis.     

未折叠蛋白应答与疾病的关系

在Ca2 稳态平衡紊乱、葡萄糖饥饿、错误折叠蛋白质的表达、蛋白质糖基化的抑制或胆固醇合成超载等胁迫条件下,会导致内质网内积累大量的未折叠蛋白质,形成内质网应激(endoplasmic reticulum stress,ERS),对细胞产生根本性的危害。在应激条件下,内质网会产生未折叠蛋白应答(unfolded protein responseUPR),通过改变细胞的转录和翻译过程来缓解内质网应激,维持细胞功能;但是,如果细胞长时间处于UPR条件下,则会诱导细胞凋亡。     

Signaling Pathways in Murine Dendritic Cells That Regulate the Response to Vesicular Stomatitis Virus Vectors That Express Flagellin

Vesicular stomatitis virus (VSV) vectors that express heterologous antigens have shown promise as vaccines in preclinical studies. The efficacy of VSV-based vaccines can be improved by engineering vectors that enhance innate immune responses. We previously generated a VSV vaccine vector that incorporates two enhancing strategies: an M protein mutation (M51R) that prevents the virus from suppressing host antiviral responses and a gene encoding bacterial flagellin (M51R-F vector). The rationale was that intracellular expression of flagellin would activate innate immune pathways in addition to those activated by virus alone. This was tested with dendritic cells (DCs) from mice containing deletions in key signaling molecules. Infection of DC with either M51R or M51R-F vector induced the production of interleukin-12 (IL-12) and IL-6 and increased surface expression of T cell costimulatory molecules. These responses were dramatically reduced in DCs from IPS-1^−/− mice. Infection with M51R-F vector also induced the production of IL-1β. In addition, in approximately half of the DCs, M51R-F vector induced pyroptosis, a proinflammatory-type of cell death. These responses to flagellin were ablated in DCs from NLRC4^−/− mice but not Toll-like receptor 5-deficient (TLR5^−/−) mice, indicating that they resulted from inflammasome activation. These results demonstrate that flagellin induces additional innate immune mechanisms over those induced by VSV alone.     

Physical and Genetic Interaction between Ammonium Transporters and the Signaling Protein Rho1 in the Plant Pathogen Ustilago maydis

Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those of Saccharomyces cerevisiae and Ustilago maydis) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen U. maydis, we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactions in vivo. This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interaction in vivo of an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis in U. maydis.     

Unfolded Protein Response Signaling and Metabolic Diseases

The endoplasmic reticulum (ER) is a central organelle for protein biosynthesis, folding, and traffic. Perturbations in ER homeostasis create a condition termed ER stress and lead to activation of the complex signaling cascade called the unfolded protein response (UPR). Recent studies have documented that the UPR coordinates multiple signaling pathways and controls various physiologies in cells and the whole organism. Furthermore, unresolved ER stress has been implicated in a variety of metabolic disorders, such as obesity and type 2 diabetes. Therefore, intervening in ER stress and modulating signaling components of the UPR would provide promising therapeutics for the treatment of human metabolic diseases.     

未折叠蛋白反应在胰腺癌发生发展中的作用及意义

胰腺癌是一种致死率相当高的消化系统肿瘤，其起病隐蔽导致早期诊断困难。近期研究发现，内质网应激 (endoplasmic reticulum stress，ERS) 状态下的未折叠蛋白反应 (unfolded protein response，UPR) 通路的调节作用，对于胰腺癌发生发展至关重要。UPR通路伴侣蛋白 GRP78 抑制了胰腺导管腺癌 (pancreatic adenocarcinoma，PDAC)细胞的凋亡，并增强了其化学抗性和耐药性。而 UPR 途径及其调节因子对于血管内皮生长因子 (vascular endothelial growth factor，VEGF) 的调节作用，有助于胰腺癌抵抗缺血缺氧环境。尝试靶向 UPR 途径关键调节因子的药物来控制胰腺癌的研究，可以为胰腺癌的治疗开辟新的途径。本文通过对近年来 UPR 在胰腺癌发生发展中的作用及意义进行综述，希望为通过调控 UPR 通路作为针对治疗胰腺癌的关键过程的一种新型抗癌方法研究提供参考。     

未折叠蛋白反应在胰腺癌发生发展中的作用及意义

胰腺癌是一种致死率相当高的消化系统肿瘤,其起病隐蔽导致早期诊断困难。近期研究发现,内质网应激 (endoplasmic reticulum stress,ERS) 状态下的未折叠蛋白反应 (unfolded protein response,UPR) 通路的调节作用,对于胰腺癌发生发展至关重要。UPR通路伴侣蛋白 GRP78 抑制了胰腺导管腺癌 (pancreatic adenocarcinoma,PDAC)细胞的凋亡,并增强了其化学抗性和耐药性。而 UPR 途径及其调节因子对于血管内皮生长因子 (vascular endothelial growth factor,VEGF) 的调节作用,有助于胰腺癌抵抗缺血缺氧环境。尝试靶向 UPR 途径关键调节因子的药物来控制胰腺癌的研究,可以为胰腺癌的治疗开辟新的途径。本文通过对近年来 UPR 在胰腺癌发生发展中的作用及意义进行综述,希望为通过调控 UPR 通路作为针对治疗胰腺癌的关键过程的一种新型抗癌方法研究提供参考。     

Endocytosis in the plant-pathogenic fungus Ustilago maydis

Summary. Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi. Correspondence and reprints: MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Federal Republic of Germany.     

SirT3 Regulates the Mitochondrial Unfolded Protein Response

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPR^mt) has been reported recently. So far, the UPR^mt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPR^mt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.     

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.