clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis sugge... more clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase E sigma A. The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner. This is the first evidence that sigma B in B. subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins. PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.
The immunodominant MART-126(27)-35 epitope, liberated from the differentiation antigen MART-1/Mel... more The immunodominant MART-126(27)-35 epitope, liberated from the differentiation antigen MART-1/Melan-A, has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 or β1i/LMP2 and β5i/LMP7 interfere with MART-126-35 epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1, proteasome activator PA28 and ER-resident aminopeptidase ERAP1 impair MART-126-35 epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-126-35 epitope by over-trimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-126-35 epitope generation even in the absence of IFN-γ. In summa...
Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tu... more Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through ...
The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, wa... more The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.
The proteasome is a multi-catalytic protein complex whose primary function is the degradation of ... more The proteasome is a multi-catalytic protein complex whose primary function is the degradation of abnormal or foreign proteins. Upon exposure of cells to interferons (IFNs), the β1i/LMP2, β2i/MECL-1, and β5i/LMP7 subunits are induced and incorporated into newly synthesized immunoproteasomes (IP), which are thought to function solely as critical players in the optimization of the CD8(+) T-cell response. However, the observation that IP are present in several non-immune tissues under normal conditions and/or following pathological events militates against the view that its role is limited to MHC class I presentation. In support of this concept, the recent use of genetic models deficient for β1i/LMP2, β2i/MECL-1, or β5i/LMP7 has uncovered unanticipated functions for IP in innate immunity and non-immune processes. Herein, we review recent data in an attempt to clarify the role of IP beyond MHC class I epitope presentation with emphasis on its involvement in the regulation of protein homeostasis, cell proliferation, and cytokine gene expression.
clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis sugge... more clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase E sigma A. The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner. This is the first evidence that sigma B in B. subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins. PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.
The immunodominant MART-126(27)-35 epitope, liberated from the differentiation antigen MART-1/Mel... more The immunodominant MART-126(27)-35 epitope, liberated from the differentiation antigen MART-1/Melan-A, has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 or β1i/LMP2 and β5i/LMP7 interfere with MART-126-35 epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1, proteasome activator PA28 and ER-resident aminopeptidase ERAP1 impair MART-126-35 epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-126-35 epitope by over-trimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-126-35 epitope generation even in the absence of IFN-γ. In summa...
Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tu... more Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through ...
The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, wa... more The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.
The proteasome is a multi-catalytic protein complex whose primary function is the degradation of ... more The proteasome is a multi-catalytic protein complex whose primary function is the degradation of abnormal or foreign proteins. Upon exposure of cells to interferons (IFNs), the β1i/LMP2, β2i/MECL-1, and β5i/LMP7 subunits are induced and incorporated into newly synthesized immunoproteasomes (IP), which are thought to function solely as critical players in the optimization of the CD8(+) T-cell response. However, the observation that IP are present in several non-immune tissues under normal conditions and/or following pathological events militates against the view that its role is limited to MHC class I presentation. In support of this concept, the recent use of genetic models deficient for β1i/LMP2, β2i/MECL-1, or β5i/LMP7 has uncovered unanticipated functions for IP in innate immunity and non-immune processes. Herein, we review recent data in an attempt to clarify the role of IP beyond MHC class I epitope presentation with emphasis on its involvement in the regulation of protein homeostasis, cell proliferation, and cytokine gene expression.
Uploads