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B Chaudhuri

Publications and source records attributed to B Chaudhuri.

At least 37 records · Page 2Linked to original sources
PubMed1998

The p16(INK4A) protein and flavopiridol restore yeast cell growth inhibited by Cdk4.

Cyclin-dependent kinase 4 (Cdk4) activity is misregulated in most cancers. Loss of Cdk4 regulation can occur through overexpression of Cdk4 catalytic subunit or its regulatory partner cyclin D1, or if the Cdk4-specific inhibitory protein p16(INK4A) is inactive. We have attempted to express the two human subunits, Cdk4 and cyclin D1, in the yeast Saccharomyces cerevisiae. Surprisingly, expression of Cdk4 alone, under control of the strong GAL promoter, inhibits cell growth. Coexpression of both subunits allows formation of an active Cdk4-cyclin D1 complex which accentuates growth arrest. In cells expressing Cdk4 only, growth is restored by overexpressing human Cdc37, a Cdk4-binding molecular chaperone. Interestingly, the effect of Cdk4 on yeast is also overcome by both p16- and p21-families of Cdk-inhibitory proteins. Moreover, flavopiridol, a compound which inhibits Cdk4 enzyme activity, restores cell division. The fact that p16(INK4A) and flavopiridol negate Cdk4-mediated suppression of yeast cell growth implies that this simple system can be used as a screen for identifying Cdk4-specific antagonists which may mimic p16(INK4A) in the cancer cell cycle.

Cyclin D1↗
PubMed1997

Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.

The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected. Coexpression of Bcl-xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

Apoptosis↗
PubMed1997

apd1+, a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump.

Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1+ was found to encode the first enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase, gcs1+. This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase gsh2+, also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe.

ATP-Binding Cassette Transporters↗
PubMed1996

Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae.

In mammalian cells, the Bcl-2 and Bcl-x(L) proteins suppress programmed cell death whereas the topographically similar Bax protein accelerates the apoptotic process. Recently published data suggest that expression of the human Bax-alpha gene is lethal for the yeast Saccharomyces cerevisiae and that this toxicity can be overcome by co-expressing Bcl-2 or Bcl-x(L). Our findings corroborate these results. However, we find that although Bax induction invariably stops cell growth under all circumstances, it does not lead to death in 'petite' cells. Petites cannot respire because they lack functional mitochondria. It seems that in 'grande' cells, which do possess normal mitochondrial DNA, nutritional limitation is critical for increased mortality. Surprisingly, murine Bcl-2 lacking the membrane anchor of human Bcl-2 has no effect on grande cells, but can efficiently rescue petites in rich medium. It has been suggested that the C-terminal membrane anchor of human Bcl-2 may have a crucial role in rescuing apoptosis in mammalian cells. When murine Bcl-2 is fused to the membrane anchor of yeast mitochondrial Mas70 protein, the Bcl-2 variant mBcl-2-mma rescues not only petites but also grandes, just like human Bcl-x(L). The rescuing ability of Bcl-x(L), which contains its own membrane anchor, surpasses that of mBcl-2-mma. Our results indicate that the process involving Bax-induced growth inhibition followed by possible lethality, and the rescuing effect of Bcl-2 and Bcl-x(L) is linked to yeast mitochondrial function. We propose a model which is consistent with these observations.

Amino Acid Sequence↗
PubMed1995

The neuroendocrine protein 7B2 contains unusually potent transcriptional activating sequences.

The expression of the 7B2 protein, secreted from a variety of neural and endocrine tissues, increases dramatically in specific neuroendocrine tumors. We have recently shown that human 7B2 can act as a molecular chaperone in the deaggregation of proteins in vitro. In order to identify polypeptides which might bind 7B2 in vivo, the yeast two-hybrid system was employed. Surprisingly, mere covalent linkage of 7B2 to the DNA-binding domains of two yeast transcription activators, Ace1 and Gal4, activates transcription from the ACE1 and GAL4 operon. 7B2's ability to activate nuclear transcription surpasses that of Ace1 and compares favourably with the strong activation domain of the tumor suppressor protein, p53. Our results suggest that 7B2 must possess an activating sequence, a domain which defines all transcriptional activator proteins. Like the acidic activation domains of some transcriptional activators, 7B2 also binds the yeast TATA-box binding protein, an essential polypeptide in the basic transcription machinery. Deletion analysis of the gene encoding 7B2 reveals two independent transcriptional activating sequences in the 185 amino acid protein. It is therefore conceivable that 7B2 not only has a functional role in the secretory pathway but also in the nucleus. Moreover, these findings raise an intriguing question regarding the activation domains of 7B2 and their possible link to 7B2's oncogenic potential.

Amino Acid Sequence↗
PubMed1995

Only in the presence of immunophilins can cyclosporin and FK506 disrupt in vivo binding of calcineurin A to its autoinhibitory domain yet strengthen interaction between calcineurin A and B subunits.

The two immunosuppressants cyclosporin A (CsA) and FK506 exert their major therapeutic effect by inhibiting T-cell activation. It is believed that the drugs first bind to their cellular receptors, known as immunophilins, and then target the protein phosphatase calcineurin for inhibition. The catalytic activity of calcineurin is regulated by its autoinhibitory domain (AID) and by the calcium-binding proteins calcineurin B (CnB) and calmodulin. We have used the yeast two-hybrid system to show that AID, CnB and calmodulin can only bind to a truncated catalytic subunit of yeast calcineurin (i.e.,CnA1 delta), devoid of AID, but not to full-length CnA1. Both CsA and FK506 cause disruption of the CnA1 delta-AID interaction, whereas their presence permits CnA1 delta to bind more strongly to CnB. In contrast, the binding of CnA1 delta to calmodulin is unaffected by the immunosuppressants. Significantly, in the absence of its cognate cytosolic receptor, neither CsA nor FK506 inhibits or stimulates the CnA1 delta-AID, CnA1 delta-CnB interactions. These in vivo observations not only provide supportive evidence for the mechanism by which drug-receptor complexes could modulate calcineurin activity but also unveil the possibility of identifying novel immunophilin-independent calcineurin inhibitors which may disturb the association of CnA1 delta to AID.

Base Sequence↗
PubMed1995

The neuroendocrine protein 7B2 acts as a molecular chaperone in the in vitro folding of human insulin-like growth factor-1 secreted from yeast.

The neuroendocrine protein 7B2 prevents premature activation of PC2, an enzyme involved in the processing of prohormones in the secretory pathway. We inquired if this chaperone-like function encompasses a broader role for 7B2 in the folding of hormone-like proteins. As a test, the fate of misfolded human insulin-like growth factor-1 (IGF1) was assessed, in the presence and absence of 7B2. Most of the recombinant IGF1 molecules, secreted from yeast, are a conglomeration of inactive multimers which are either disulfide-linked or mere physical aggregates. We find that yeast-produced 7B2 influences the in vitro conversion of inactive molecules into active monomers. However, the amounts of disulfide-linked dimers remain unaffected during this conversion. Interestingly, both 7B2 and the molecular chaperone DnaK interact with IGF1 in the yeast two-hybrid system. Like DnaK, 7B2 also binds the tumor suppressor protein p53. Binding of DnaK to exposed epitopes of aggregated proteins is known to be a prerequisite for deaggregation. It is conceivable that 7B2 participates in an analogous manner in the dissociation of non-covalently linked multimers of IGF1. Our results indicate that 7B2 might find an application in the deaggregation of potentially useful therapeutic proteins.

Base Sequence↗
PubMed1995

A C-terminal domain, which prevents secretion of the neuroendocrine protein 7B2 in Saccharomyces cerevisiae, inhibits Kex2 yet is processed by the Yap3 protease.

Recent reports reveal that the C-terminal half of the neuroendocrine polypeptide 7B2 selectively inhibits and binds PC2, a mammalian prohormone converting enzyme that is homologous to the yeast pro-alpha-factor processing protease Kex2. During attempted secretion of the 185 amino-acid human 7B2 in Saccharomyces cerevisiae, we observe that the protein is mostly retained inside the cell. However a mutant polypeptide (7B2 delta 1), where the C-terminal 48 amino acids of 7B2 are deleted, is efficiently secreted. Two shorter C-terminal truncations either permit poor secretion or no secretion at all. Surprisingly, full-length 7B2 but not 7B2 delta 1 abolishes the catalytic activity of Kex2, indicating that C-terminal residues of 7B2 might also be important for inhibition of the yeast protease. When the KEX2 gene is disrupted, yeast cells unexpectedly secrete a 7B2 variant similar in size to 7B2 delta 1, suggesting involvement of the alternate yeast prohormone convertase Yap3 in processing. Secretion is enhanced by overexpression of Yap3 and by the presence of a Lys-Arg residue at the processing site of precursor 7B2. These results purport that, in neuroendocrine cells too, secretion of 7B2 could be mediated by a homologue of Yap3.

Aspartic Acid Endopeptidases↗
PubMed1995

The unfolded-protein-response element discriminates misfolding induced by different mutant pro-sequences of yeast carboxypeptidase Y.

The C-terminal region of the chaperone-like pro-sequence (py) of yeast carboxypeptidase Y (CPY) is suggested to be crucial for the folding of mature CPY. In order to study the influence of hydrophobic residues in this domain, a set of mutations have been introduced in py. Unexpectedly, only small amounts of CPY precursors are expressed when Leu108, at the C-terminal end of py, is substituted for polar residues Lys, Arg or Asp. In contrast, substitution with hydrophobic residues Val, Ile or Ala permit normal expression. Interestingly, the poorly expressed molecules are coreglycosylated, implying that they have failed to leave the endoplasmic reticulum (ER). The ER-retained molecules caused an induction in the levels of BiP, signifying that polar substitutions at position 108 of pre-py-CPY induce misfolding. Quite surprisingly, a reporter gene, linked to concatamerized unfolded-protein-response elements, reveals that py-mediated misfolding of CPY is not really identical in all mutants. This shows that a simple transcriptional assay can assess the subtleties of pro-sequence mediated protein folding in an eukaryotic cell.

Base Sequence↗
PubMed1995

The interaction between the catalytic A subunit of calcineurin and its autoinhibitory domain, in the yeast two-hybrid system, is disrupted by cyclosporin A and FK506.

The Ca(2+)-calmodulin dependent protein phosphatase, calcineurin, is thought to mediate the action of the two immunosuppressants, cyclosporin A (CsA) and FK506. Calcineurin from all species consists of a catalytic A subunit and a regulatory peptide B, which plays an essential role in catalysis. The enzymatic function is probably also regulated by an autoinhibitory domain (AID) present in the catalytic subunit. We have used the yeast two-hybrid system to show that the putative AID of the yeast catalytic subunit Cna1 binds only to truncated Cna1, devoid of AID. Although deletion of the genes encoding the yeast catalytic subunits of calcineurin (CNA1 and CNA2) maintain the interaction, absence of the regulatory subunit Cnb1 prevents binding. Interestingly, both CsA and FK506 disrupt this interaction, whereas binding of Cna1 to calmodulin remains unaffected. This indicates that a simple cellular system, developed in yeast, could provide further insight into an understanding of calcineurin inhibition.

Amino Acid Sequence↗
PubMed1993

Isolation, characterization, and mapping of temperature-sensitive mutations in the genes essential for lysogenic and lytic growth of the mycobacteriophage L1.

Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42 degrees but not at 32 degrees while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42 degrees, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.

Chromosome Mapping↗
PubMed1993

Distribution of estrogen receptor in ductal carcinoma in situ of the breast.

BACKGROUND: Long-term antiestrogen therapy has been suggested as a possible treatment alternative for ductal carcinoma in situ (DCIS) of the breast. However, very little information is available on the distribution of estrogen receptor (ER) and treatment success with antiestrogen for such lesions. METHODS: Thirty-two formalin-fixed tissue specimens of DCIS from 32 female patients aged 38 to 71 years were evaluated for the presence of ER by an immunoperoxidase technique. Antigenic sites for ER were exposed by treating the tissue sections with deoxyribonuclease followed by peroxidase-antiperoxidase staining with monoclonal antibody against ER. Parallel negative controls were run with negative control monoclonal antibody and normal rat serum. The quality control for positive staining was performed with tissue sections from specimens with known ER detected by the radioreceptor method. RESULTS: Eighteen (60%) of the 32 lesions were positive for ER. In 7 of the 18 lesions less than 25% of cells stained positive for ER and in 4 of the 18 more than 50% of cells stained positive for ER. CONCLUSIONS: The incidence of ER in DCIS is similar to the incidence of ER in invasive carcinoma, leading to the speculation that ER-positive invasive carcinoma originates from an ER-positive precursor lesion. Because only 60% of the cases have detectable ER and approximately 70% of positively stained ERs are expected to be functional (as in invasive carcinoma), it appears that approximately 42% of the patient population with DCIS will benefit from antiestrogen therapy.

Adenocarcinoma↗
PubMed1992

A mutant Kex2 enzyme with a C-terminal HDEL sequence releases correctly folded human insulin-like growth factor-1 from a precursor accumulated in the yeast endoplasmic reticulum.

Mutations in the pro region of the yeast DNA hybrid of prepro-alpha-factor and human insulin-like growth factor-1 (IGF-1) cause the accumulation, in the yeast Saccharomyces cerevisiae, of an unglycosylated precursor protein where the pre sequence is missing. The prepro sequence of the prepro-alpha-factor consists of a pre or signal sequence and a proregion which possesses three sites for N-glycosylation. Isolation of a precursor, where the pro region is still linked to IGF-1 through a pair of dibasic amino acid residues, implies that the polypeptide may have translocated into the endoplasmic reticulum (ER) but has not been processed by the Golgi membrane-bound Kex2 endoprotease. However, the lack of any N-glycosylation in the translocated polypeptide is surprising. The mutated pro region, can be processed, in vitro, by treatment with a soluble form of the Kex2 enzyme. It is also possible to release the pro region, in vivo, by coexpressing a mutant Kex2 protease which is partially retained in the ER with the help of the C-terminal tetrapeptide sequence, HDEL. The mature IGF-1, which is secreted from the intracellular pool of precursor proteins, is predominantly an active, monomeric molecule, corroborating observations that early removal of the pro region before folding in the ER helps to prevent aberrant intermolecular disulfide-bond formation in IGF-1. These results have revealed the utility of the ER-retained Kex2 enzyme as a novel in vivo biochemical tool.

Amino Acid Sequence↗
PubMed1992

The pro-region of the yeast prepro-alpha-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo.

Four yeast secretion signals, the 19-amino-acid invertase signal sequence, the 17-amino-acid acid-phosphatase signal sequence, and the pre-sequence and prepro-sequence of prepro-alpha-factor have been used to look for the secretion of recombinant human insulin-like growth factor 1 (IGF1) from Saccharomyces cerevisiae. Only the prepro-sequence, often referred to as the alpha-factor leader and consisting of an N-terminal 19-amino-acid pre-sequence or signal sequence attached to a 66-amino-acid pro-region, permits secretion of IGF1. The signal sequences alone do not allow the translocation of IGF1 into the endoplasmic reticulum. This is evident from the fact that IGF1-like molecules, to which the signal sequences are still attached, accumulate intracellularly in the cytosol. Fusion of the pro-region of the alpha-factor leader to the C-terminus of the acid-phosphatase and invertase signal sequences allows IGF1 to be secreted once again. These results reveal the essential role of the pro-region of the alpha-factor leader in the secretion of IGF1 and indicate that it may have a function in guiding a nascent IGF1 polypeptide to a state in which translocation can occur.

Amino Acid Sequence↗
PubMed1992

A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide-linked dimerisation of recombinant human insulin-like growth factor-1 secreted from yeast.

The majority of the recombinant human insulin-like growth factor-1 (IGF1) molecules, secreted from yeast using the prepro sequence of the prepro-alpha-factor, are not active monomers but inactive, disulfide-linked dimers. The prepro sequence of the prepro-alpha-factor, usually referred to as the alpha-factor leader (alpha FL), consists of a pre or signal sequence and a proregion. After signal sequence removal during translocation into the endoplasmic reticulum (ER) the proregion is still attached to IGF1 when it folds to acquire a tertiary structure. Mature IGF1 is released only in a late Golgi compartment by the membrane-bound endoprotease Kex2p. We find that co-expression of a novel ER-retained Kex2p variant, soluble Kex2pHDEL, can prevent intermolecular disulfide bond formation between two IGF1 molecules, implying that the presence of the proregion during the folding of IGF1 in the ER could be a reason for disulfide-linked dimerisation. This result indicates that the proregion of the alpha FL may have a role in the folding of some heterologous proteins in yeast, and that the ER-retained Kex2p mutant could be used as a convenient tool to study the cellular function of the proregions present naturally in various eucaryotic precursor proteins.

Blotting, Western↗
PubMed1992

A novel Kex2 enzyme can process the proregion of the yeast alpha-factor leader in the endoplasmic reticulum instead of in the Golgi.

The prepro sequence of the yeast prepro-alpha-factor, usually referred to as the alpha-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae. The alpha-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the alpha-factor leader--human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins.

Base Sequence↗