Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and Cyp40

Large peptidyl-prolyl cis/trans isomerases (PPIases) are important components of the Hsp90 chaperone complex. In mammalian cells, either Cyp40, FKBP51 or FKBP52 is incorporated into these complexes. It has been suggested that members of this protein family exhibit both prolyl isomerase and chaperone activity. Here we define the structural and functional properties of the three mammalian large PPIases. We find that in all cases two PPIase monomers bind to an Hsp90 dimer. However, the affinities of the PPIases are different with FKBP52 exhibiting the strongest interaction and Cyp40 the weakest. Furthermore, in the mammalian system, in contrast to the yeast system, the catalytic activity of prolyl isomerization corresponds well to that of the respective small PPIases. Interestingly, Cyp40 and FKBP51 are the more potent chaperones. Thus, it seems that both the affinity for Hsp90 and the differences in their chaperone properties, which may reflect their interaction with the non-native protein in the Hsp90 complex, are critical for the selective incorporation of a specific large PPIase.     

Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.     

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.     

Astrocytic but not neuronal increased expression and redistribution of parkin during unfolded protein stress

Parkin is a ubiquitin ligase that facilitates proteasomal protein degradation and is involved in a common autosomal recessive form of Parkinson's disease. Its expression is part of the unfolded protein response in cell lines where its overexpression protects against unfolded protein stress. How parkin expression is regulated in brain primary cells under stress situations is however, less well established. Here, the cellular and subcellular localization of parkin under basal conditions and during unfolded protein stress was investigated in primary cultures of rat astrocytes and hippocampal neurons. Immunofluorescense microscopy and biochemical analysis demonstrated that parkin is mainly associated with the endoplasmic reticulum (ER) in hippocampal neurons while it is associated with Golgi membranes, the nuclei and light vesicles in astrocytes. The constitutive parkin expression was high in neurons as compared with astrocytes. However, unfolded protein stress elicited a selective increase in astrocytic parkin expression and a change in distribution, whereas neuronal parkin remained largely unmodified. The cell specific differences argue in favour of different cellular binding sites and substrates for the protein and a pathogenic role for astrocytes in Parkinson's disease caused by parkin dysfunction.     

SUMOylation of HNRNPA2B1 modulates RPA dynamics during unperturbed replication and genotoxic stress responses

1. Download : Download high-res image (190KB)
2. Download : Download full-size image

     

Oxidative stress‐induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson''s disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4‐TRPML1‐ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca²⁺ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4‐TRPML1‐ALG2 pathway was also activated by H₂O₂, indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation‐induced lysosomal retrograde transport involved both the TMEM55B‐JIP4 and TRPML1‐ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy‐inducing signal.     

AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis

In nature, plants are subject to changes of tempera-ture. Thus, like other organisms, plants have evolved strategies for preventing damage caused by rapid changes in temperature and for repairing what damage is unavoidable. Heat stress responses have been well documented in a wide range of organisms. In all spe-cies studied, the heat shock (HS) response is charac-terized by a rapid production and a transient accumu-lation of specific families of proteins known as heat shock proteins (Hsps) th…     

FoxO4 negatively modulates USP10 transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation-induced cardiomyocytes by regulating the Hippo/YAP pathway

Journal of Bioenergetics and Biomembranes - Acute myocardial infarction (AMI) is the main cause of death in the whole world. This study aimed to investigate whether forkhead box O4 (FoxO4) could...     

结核分枝杆菌PE＿PGRS15调控分枝杆菌细胞包膜结构与耐药性

【目的】研究结核分枝杆菌PE＿PGRS15的功能。【方法】构建过表达PE＿PGRS15蛋白的重组耻垢分枝菌酸杆形菌,通过细胞分级分离实验检测其细胞定位。通过涂布实验、扫描电镜和透射电镜观察细菌菌落形态、细菌表面形态及细胞包膜(cell envelope)结构。通过杀菌曲线法及微量肉汤稀释法检测重组菌对环境压力及抗生素的耐受性。通过染料摄取实验检测重组菌细胞壁通透性,并用气相色谱-质谱联用仪检测重组菌细胞壁脂肪酸谱。通过蛋白截短及融合实验分析PE＿PGRS15蛋白结构域的功能。【结果】PE＿PGRS15蛋白定位于重组菌细胞壁,其表达影响重组菌菌落形态和细胞包膜结构,增强重组菌对环境压力和抗生素的耐受。PE＿PGRS15的表达导致重组菌细胞包膜脂肪酸含量增加,也降低了重组菌的细胞壁通透性。PE＿PGRS15蛋白的PE结构域负责将该蛋白转运到细胞表面,而PGRS结构域介导重组菌对压力条件和抗生素的耐受。【结论】PE＿PGRS15蛋白可能通过调控耻垢分枝菌酸杆形菌细胞包膜的结构进而影响细菌菌落形态、细胞壁通透性及耐药性,为解析PE/PPE家族蛋白的功能奠定了一定的基础。     

Regulation of Bcl-2 protein expression during oxidative stress in neuronal and in endothelial cells.

The relationship between oxidative stress and Bcl-2 expression was investigated in two different experimental models of oxidative stress. Acute oxidative stress was assessed by measuring, with fluorescence microscopy and cytofluorimetry, the increase in fluorescence of the oxidation-sensitive probe dihydrorhodamine 123, both in retinal rod receptor cells exposed to bright light (0.32 mW/cm(2) for 15 minutes) and in human endothelial cells treated with the immunosuppressant cyclosporin A (200 microM for 21 h). In both cell types, acute oxidative stress reduced Bcl-2 expression and also caused a significant increase in the level of nucleosomes. Interestingly, chronic treatment with clinical concentrations of cyclosporin A (0.5-2.5 microM for 8 days) led to a significant increase in Bcl-2 expression, while nucleosomes were similar to control level. This suggests that up-regulation of Bcl-2 protein by low levels of oxidants may represent a critical factor in cellular adaptation to drug toxicity.     

Distinct reading of different structural determinants modulates the dileucine-mediated transport steps of the lysosomal membrane protein LIMPII and the insulin-sensitive glucose transporter GLUT4

Leucine-based motifs mediate the sorting of membrane proteins at such cellular sites as the trans-Golgi network, endosomes, and plasma membrane. A Leu paired with a second Leu, Ile, or Met, while itself lacking the ability to mediate transport, is the key structural feature in these motifs. Here we have studied the structural differences between the leucine-based motifs contained in the COOH tails of LIMPII and GLUT4, two membrane proteins that are transported through the secretory pathway and are targeted to lysosomes () and to a perinuclear compartment adjacent to the Golgi complex (), respectively. LIMPII and GLUT4 display negatively (Asp(470)/Glu(471)) and positively (Arg(484)/Arg(485)) charged residues, respectively, at positions -4 and -5 upstream from the critical Leu residue. The change in the charge sign of residues -4 and -5 results in missorting of LIMPII and GLUT4. We note that the acidic Glu residue at position -4 is critical for efficient intracellular sorting of LIMPII to lysosomes, but is dispensable for its surface internalization by endocytosis. Efficient intracellular sorting and endocytosis of GLUT4 require an Arg pair between positions -4 and -7. These results are consistent with the existence of distinct leucine-based motifs and provide evidence of their different readings at different cellular sites.