Unconventional secretion of α-Crystallin B requires the Autophagic pathway and is controlled by phosphorylation of its serine 59 residue.

α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.

Adenosine plasma level correlates with homocysteine and uric acid concentrations in patients with coronary artery disease.

The role of hyperhomocysteinemia in coronary artery disease (CAD) patients remains unclear. The present study evaluated the relationship between homocysteine (HCys), adenosine plasma concentration (APC), plasma uric acid, and CAD severity evaluated using the SYNTAX score. We also evaluated in vitro the influence of adenosine on HCys production by hepatoma cultured cells (HuH7). Seventy-eight patients (mean age ± SD: 66.3 ± 11.3; mean SYNTAX score: 19.9 ± 12.3) and 30 healthy subjects (mean age: 61 ± 13) were included. We incubated HuH7 cells with increasing concentrations of adenosine and addressed the effect on HCys level in cell culture supernatant. Patients vs. controls had higher APC (0.82 ± 0.5 µmol/L vs 0.53 ± 0.14 µmol/L; p < 0.01), HCys (15 ± 7.6 µmol/L vs 6.8 ± 3 µmol/L, p < 0.0001), and uric acid (242.6 ± 97 vs 202 ± 59, p < 0.05) levels. APC was correlated with HCys and uric acid concentrations in patients (Pearson's R = 0.65 and 0.52; p < 0.0001, respectively). The SYNTAX score was correlated with HCys concentration. Adenosine induced a time- and dose-dependent increase in HCys in cell culture. Our data suggest that high APC is associated with HCys and uric acid concentrations in CAD patients. Whether the increased APC participates in atherosclerosis or, conversely, is part of a protective regulation process needs further investigations.

Assembly of HCV E1 and E2 glycoproteins into coronavirus VLPs.

Hepatitis C virus (HCV) is believed to assemble by budding into membranes of the early secretory pathway, consistent with the membrane location where the viral envelope glycoproteins E1 and E2 accumulate when expressed. Coronavirus assembly also takes place at pre-Golgi membranes. Here, we generated coronavirus-like particles carrying in their envelope chimeric HCV glycoproteins composed of the ectodomains of E1 and E2, each fused to the transmembrane plus endodomain of the mouse hepatitis coronavirus spike glycoprotein. The chimeric particle system will enable structural and functional studies of the HCV glycoproteins.

Regression of left ventricular hypertrophy and improvement of diastolic function in hypertensive patients treated with telmisartan.

OBJECTIVES: The study was designed to test whether or not the angiotensin II receptor blocker telmisartan brings about regression of left ventricular (LV) concentric hypertrophy and whether or not these changes are associated with improved diastolic filling. METHODS: An echocardiographic follow-up study was performed in 85 hypertensive patients (systolic blood pressure [SBP] >140 mmHg, diastolic blood pressure [DBP] >90 mmHg) and mild-to-moderate LV hypertrophy (LV mass index related to body surface area [LVMI] 117-150 g/m2 for men and 105-150 g/m2 for women) treated with telmisartan monotherapy 40-80 mg once daily for 1 year. Blood pressure, LVMI, left atrial (LA) volumes, and diastolic function were determined at baseline and after 3, 6, 9, and 12 months of treatment. Blood pressure was also monitored at all visits. Diastolic function was assessed by examination of transmitral inflow and pulmonary vein flow patterns. RESULTS: Telmisartan reduced blood pressure; after 12 months, the mean+/-S.D. SBP and DBP were reduced from 144+/-10 to 126+/-8 mmHg (p<0.001) and from 98+/-8 to 86+/-7 mmHg (p<0.001), respectively. The LVMI was decreased from 119+/-7 to 109+/-3 g/m2 (p<0.001) after 12 months' telmisartan treatment. All patients had diastolic dysfunction at baseline. After 12 months' telmisartan treatment, a normal pattern of transmitral inflow was present in 21% of patients. The regression of LV hypertrophy observed after 12 months was associated with increased peak early diastolic velocity/peak late diastolic velocity ratio from 0.60+/-0.18 to 0.83+/-0.20 (p<0.001), shortened isovolumic relaxation time (IVRT) from 110+/-13 to 105+/-13 ms (p<0.001), and decreased deceleration time from 229+/-30 to 215+/-28 ms (p=0.002). Univariate analysis showed that shortened IVRT was related to a reduction in the LVMI and LA maximal and minimal volumes. In the multivariate analysis, the reduction in LVMI and the reduction in LA maximal and minimal volumes were independently associated with IVRT reduction. CONCLUSIONS: Telmisartan 40-80 mg is effective in LV hypertrophy regression in hypertensive patients. The reduction in LVMI due to telmisartan monotherapy was associated with a significant improvement of diastolic filling parameters and with a significant reduction of LA volumes.

Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents.

It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.

The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment.

There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.

Hepatitis C virus structural proteins reside in the endoplasmic reticulum as well as in the intermediate compartment/cis-Golgi complex region of stably transfected cells.

The intracellular localization of hepatitis C virus structural proteins was analyzed by confocal immunofluorescence microscopy, cell fractionation, and immunoelectron microscopy in stably transfected cells that do not overexpress the viral proteins. The results strongly suggest that at steady state the structural proteins reside not only in the endoplasmic reticulum but also in the intermediate compartment and cis-Golgi complex region. By analogy with other viral systems, this finding raises the possibility that the intermediate compartment and cis-Golgi complex play a role in the assembly and budding of hepatitis C virus.

Effects of simulated microgravity on metabolic activities related to DNA damage and repair in lymphoblastoid cells.

We adopted a simple experimental framework to follow the dependence of structural aberrations and the modifications in selected metabolic processes correlated with the exposure of cells to microgravity. Alterations to the cellular metabolism induced by exposure to microgravity are evidentiated in the modification of PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to microgravity in a Random Positioning Machine (RPM) in complete medium for 48 hours. At the end of this period a part of these cells are immediately analysed for the parameters reported above and the remaining were furtherly incubated in standard laboratory conditions to document eventual defects during the phases of the recovery process. A part of cells, just after exposure to microgravity, were also subjected to treatment with a strong damaging agent, KBrO3, and these cells were subsequently analyzed. This final treatment was meant to amplify the eventual deficiencies experienced by microgravity-exposed cells in the DNA repair process also in dependence with the alterated metabolic conditions resulting after the exposure to microgravity.

Assembly of the CD8alpha/p56(lck) protein complex in stably expressing rat epithelial cells.

We have previously characterized the biogenesis of the human CD8alpha protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8alpha/p56(lck) protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56(lck), or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8alpha, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8alpha established hetero-oligomeric complexes with p56(lck), as revealed by co-immunoprecipitation assays performed with anti-CD8alpha antibody. Moreover, p56(lck) bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56(lck) binds rapidly to CD8alpha and most of the p56(lck) is bound to CD8alpha at steady state; (2) CD8alpha/p56(lck) protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56(lck) reaches the cell surface.

Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification.

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2.

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.

Induction of apoptosis and inhibition of signalling pathways by alkylated purines.

Addition of growth factors such as EGF and insulin to serum-starved G(0) Chinese hamster fibroblast cells results in activation of the phosphatidylinositol 3-kinase (PI3-K)/p70 S6 kinase (p70(S6K)) pathway and the ras-raf mitogen-activated kinase (MAPK) pathway. Activation of these pathways is usually associated with protection of cells from apoptosis. We have studied the effect of three alkylpurines, O(6)-methylguanine (O6meG), O(6)-ethylguanine (O6etG) and 6-dimethylaminopurine (6DMAP) on two particular steps of these pathways, namely activation of p70(S6K) and of MAPK. Under the same experimental conditions we studied the ability of these alkylpurines to induce apoptosis. Our results show that the three alkylpurines induced apoptosis with increasing efficiency from O6meG to 6DMAP to O6etG. The induction of apoptosis was phase specific, with the G(0)/G(1) phase being most sensitive. A reduced apoptotic response was observed in cells with abnormal nuclear accumulation of mutant or wild-type p53, suggesting that functional p53 was required for the induction of apoptosis. At concentrations inducing apoptosis the three alkylpurines inhibited p70(S6K) activity, while they had the opposite effect on MAPK. Rapamycin, a specific inhibitor of the p70(S6K) pathway, did not induce apoptosis at doses inhibiting p70(S6K) activity, suggesting that p70(S6K) is not directly involved in apoptosis. As expected, and in line with results reported by others, wortmannin, an upstream inhibitor of the p70(S6K) pathway, did induce apoptosis. We propose that activation of the MAPK pathway and simultaneous inhibition of the p70(S6K) pathway induce an apoptotic response in the cell.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1.

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.

Micronucleus frequencies in exfoliated buccal cells in normal mucosa, precancerous lesions and squamous cell carcinoma.

OBJECTIVE: To assess the value of micronuclei in the characterization of precancerous lesions of the oral cavity with reference to their likelihood of progressing to malignant lesions. STUDY DESIGN: The frequency of micronuclei was determined in exfoliated cells from normal oral mucosa, a preneoplastic condition (leukoplakia) and precancerous lesions with and without dysplasia, squamous cell carcinomas and sites of previous carcinomas that had been removed. RESULTS: Average micronucleus frequencies were increased in precancerous lesions as compared to normal mucosa and further increased in carcinomas, suggesting that micronuclei are a biomarker of neoplastic progression in this type of cancer. With all samples, micronucleus frequencies were systematically higher when cells were collected by vigorous than by light scraping, suggesting a decreasing gradient from basal to superficial layers of mucosa. The micronucleus frequency did not vary with the sex or age of patients, while it did vary with the anatomic site of the lesions. CONCLUSION: Although the gradual increase in micronucleus counts from normal mucosa to precancerous lesions to carcinomas suggests a link of this biomarker with neoplastic progression, the large overlapping of data prevents its use as a predictor of progression of precancerous lesions to malignancy in individual patients.

Sequence and expression of the monkey homologue of the ER-golgi intermediate compartment lectin, ERGIC-53.

We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66. 98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta.

A different intracellular distribution of a single reporter protein is determined at steady state by KKXX or KDEL retrieval signals.

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.

Cell fractionation analysis of human CD8 glycoprotein transport between endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells.

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.

Reduction of genotoxic effects of MNNG by butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) is a food preservative with markedly contradictory effects. On one side many studies showed its antimutagenic and anticarcinogenic effects but on the other side dietary levels of BHA were reported to cause gastrointestinal hyperplasia in rodents. We studied the influence of BHA on cytotoxicity, mutagenicity, and DNA-damaging activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V79 cells cultured in vitro. Our results showed that BHA significantly reduced the frequency of 6-thioguanine resistant (6-TGr) mutations and micronuclei induced in V79 cells by MNNG. These antimutagenic effects of BHA were, however, accompanied by a very marked increase of MNNG toxicity and also slightly increased level of MNNG-induced DNA damage. For evaluation of toxicity we used three methods: (i) trypane blue exclusion; (ii) plating efficiency; and (iii) intensity of cellular macromolecule synthesis. The level of DNA damage was measured by the comet assay. On the basis of obtained results we suggest that BHA, which induces phase II detoxifying enzymes, probably doesn't reduce the level of DNA damage induced in time of MNNG-treatment but it reduces the level of DNA damage created during a long-term period needed for expression of 6-TGr mutations and micronuclei.