Role of HSPA1L as a cellular prion protein stabilizer in tumor progression via HIF-1α/GP78 axis.

The cellular prion protein (PrPC) is associated with metastasis, tumor progression and recurrence; however, the precise mechanisms underlying its action is not well understood. Our study found that PrPC degradation decreased tumor progression in colorectal cancer (CRC). In a CRC cell line and human CRC tissue exposed to hypoxia, induced heat-shock 70-kDa protein-1-like (HSPA1L) expression stabilized hypoxia-inducible factor-1α (HIF-1α) protein and promoted PrPC accumulation and tumorigenicity in vivo. PrPC was degraded via the proteasome pathway mediated by the ubiquitin-protein E3 ligase glycoprotein 78 (GP78), which interacts directly with PrPC. However, hypoxia-induced HSPA1L interacted with GP78 and inhibited its functions. HSPA1L knockdown facilitated the interaction of GP78 and PrPC, thereby increasing PrPC ubiquitination. Thus, GP78 was identified as the ubiquitinase for PrPC, thereby revealing an essential mechanism that controls PrPC levels in CRC. Our results suggest that the HSPA1L/HIF-1α/GP78 axis has a crucial role in PrPC accumulation during tumor progression.

Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein.

The hepatis B virus X protein (HBx) has been thought to be implicated in the development of hepatocellular carcinoma. Although many functions of HBx have been reported, it is not clear which of HBx functions is important in hepatocellular carcinogenesis. To study HBx function, we produced a monoclonal anti-HBx Ab secreted by hybridoma cell clone H7 and mapped its epitope to a region of HBx between amino acids 29 and 48 by Western blot with truncated forms of HBx and by enzyme-linked immunoadsorbent assay (ELISA) with synthetic HBx peptides. The variable regions of H7 anti-HBx Ab were cloned by polymerase chain reaction using the degenerate-primers and by the 5' rapid amplification-cDNA end method. The sequence analyses revealed that the variable gene segments of the heavy and light chains are the members of mouse heavy chain variable gene 1 family and kappa light chain variable gene 2 family, respectively. In addition, J(H)2 or Jkappa4 gene segment at the end of the heavy-chain or light-chain variable region and DSP2.x gene segment in the CDR 3 of heavy chain were identified.

G-protein coupled receptor from yeast Saccharomyces cerevisiae.

The Saccharomyces cerevisiae GPR1 (G-protein coupled receptor) gene was isolated using two-hybrid system with a heterotrimeric GTP binding protein alpha subunit Gpa2p as a bait. The GPR1 gene encodes 961 amino acids with predicted seven transmembrane segments and two large cytosolic regions as third cytosolic loop with 350 amino acids where asparagine-rich region was found and the C-terminal region with 283 amino acids. The Gpr1p interacted with Gpa2p at C-terminal region with 131 amino acid residues as well as third cytosolic loop. Disruption of the GPR1 gene was not lethal and did not affect to the cell growth. The Gpr1p-GFP fusion protein localized at the cell surface. These results suggest that Gpr1p is a G-protein coupled receptor which localized at plasma membrane. It is likely that a Gpr1p monitors the extracellular signal such as nutrition and transduce it via Gpa2p a possible positive regulator of cAMP level.

Gpr1p, a putative G-protein coupled receptor, regulates glucose-dependent cellular cAMP level in yeast Saccharomyces cerevisiae.

How cells monitor the availability of nutrition and transduce signals is a fundamental, unanswered question. We have found that Gpr1p, a recently identified G-protein (Gpa2p) coupled receptor in yeast Saccharomyces cerevisiae, regulate the cellular cAMP level in response to glucose. The glucose-induced higher cAMP level found in the strain with GPA2 in multicopy plasmid decreased by deletion of GPR1 gene. A transient increase of cAMP in response to glucose was not observed in a Deltagpr1 mutant strain and this defect was complemented and restored by introducing GPR1 gene with YCp vector. Gpr1p was also required for the increase of cAMP in response to other fermentable sugars. Both membrane proximal regions o the third cytosolic loop in Gpr1p, which has been shown to be important for coupling to G-proteins, were also required for glucose-induced transient increase of cAMP. Our findings suggest that Gpr1p is part of the nutrition sensing machinery most likely acting as a receptor to monitor glucose as well as other fermentable sugars and regulate cellular cAMP levels.

Siderophore-iron uptake in saccharomyces cerevisiae. Identification of ferrichrome and fusarinine transporters.

A family of four putative transporters (Arn1p-4p) in Saccharomyces cerevisiae is expressed under conditions of iron deprivation and is regulated by Aft1p, the major iron-dependent transcription factor in yeast. One of these, Arn3p/Sit1p, facilitates the uptake of ferrioxamine B, a siderophore of the hydroxamate class. Here we report that ARN family members facilitated the uptake of iron from the trihydroxamate siderophores ferrichrome, ferrichrome A, and triacetylfusarinine C. Uptake of siderophore-bound iron was dependent on either the high-affinity ferrous iron transport system or the ARN family of transporters. The specificity of each siderophore for individual transporters was determined. Uptake of ferrichrome and ferrichrome A was facilitated by both Arn1p and Arn3p. Uptake of triacetylfusarinine C was facilitated by Arn2p, although small amounts of uptake also occurred through Arn1p and Arn3p. In contrast to the trihydroxamates, uptake of iron from the dihydroxamate rhodotorulic acid occurred only via the high-affinity ferrous iron system. Epitope-tagged Arn1p was expressed in intracellular vesicles in a pattern that was indistinguishable from that of Arn3p, whereas Ftr1p, a component of the high-affinity ferrous system, was expressed on the plasma membrane. These data indicate that S. cerevisiae maintains two systems of siderophore uptake, only one of which is located on the plasma membrane.

The role of the FRE family of plasma membrane reductases in the uptake of siderophore-iron in Saccharomyces cerevisiae.

Saccharomyces cerevisiae takes up siderophore-bound iron through two distinct systems, one that requires siderophore transporters of the ARN family and one that requires the high affinity ferrous iron transporter on the plasma membrane. Uptake through the plasma membrane ferrous iron transporter requires that the iron first must dissociate from the siderophore and undergo reduction to the ferrous form. FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron. The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown. Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p. By indirect immunofluorescence, Fre3p was expressed on the plasma membrane in a pattern similar to that of Fet3p, a component of the high affinity ferrous transporter. Enterobactin, a catecholate siderophore, was not a substrate for Fre3p, and reductive uptake required either Fre1p or Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations. We propose that Fre3p and Fre4p are siderophore-iron reductases and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources.

GPR1 regulates filamentous growth through FLO11 in yeast Saccharomyces cerevisiae.

Cell growth and differentiation are regulated by nutrient availability in the yeast Saccharomyces cerevisiae. Under conditions of nitrogen limitation, diploid cells of S. cerevisiae differentiate to a filamentous growth known as a pseudohyphal growth, while haploid cells produce invasive filaments which penetrate the agar in nutrient-rich medium. We have found that GPR1, which encodes a putative G-protein-coupled receptor, is required for both pseudohyphal and invasive growth. Pseudohyphal growth was defective in Deltagpr1/Deltagpr1 mutant strain and this defect was reversed by addition of cAMP. Also, haploid Deltagpr1 mutant strain was defective in invasive growth. Northern blot analysis revealed that the transcriptional level of FLO11, which encodes a recently identified cell surface flocculin required for pseudohyphal growth, was reduced in Deltagpr1 mutant strain. These results indicate that GPR1 regulates both pseudohyphal and invasive growth by a cAMP-dependent mechanism.

Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake.

In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1. cDNA microarrays were used to identify new iron and AFT1-regulated genes. Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters. These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease. Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine-bound iron and growth on ferrioxamine as an iron source. The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments. Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12. In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases. These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.