- Email: [email protected]
How is the gate to the proteasome opened?
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HEADLINES
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ANNOUNCEMENT Regular readers will no doubt be familiar with TCB Headlines which provide short, insightful summaries of the latest cell-biology research papers. But did you know that you can now access timely summaries of outstanding recently published papers on a daily basis through the Commentaries section of our recently enhanced BioMedNet online service? Authored by distinguished scientists, Commentaries provide a thought-provoking synopsis of scientific endeavour across the life sciences, including of course cell biology. Access to Commentaries is FREE. All you need do to find the Commentaries section of BioMedNet is go to http://www.bmn.com, click on the ‘News and Comment’ link and then press the Commentaries button (you might need to register as a BioMedNet member if you haven’t already). Selected commentaries may also appear subsequently as Headlines in your TCB magazine, and collectively Headlines will be archived electronically in the ‘Journal Club’ section of BioMedNet. As a further commitment to providing value for you our readers, we hope you appreciate the new look and feel of this month’s bigger TCB issue. As part of a programme of continuous investment in the Trends journals, which will unfold over the coming months, we will provide you more reviews, further cutting-edge analysis and thoughtful opinion articles; indeed – more essential coverage than ever before of your science. We welcome your comments and suggestions on these developments – contact the Editor at [email protected]
Hot plants, hot yeast Members of the heat-shock protein 70 (Hsp70) family act as central players when vertebrates and flies acquire tolerance to otherwise-lethal temperatures. Bacteria and yeast, by contrast, use the Hsp100 family in this role. The Vierling1,2 and Lindquist2 labs now place plants squarely in the microbial camp. In a direct assault on the problem, Queitch et al.2 generated transgenic Arabidopsis plants that underexpressed (by antisense and cosuppression methods) or constitutively accumulated Hsp101. Low-expressing plants lost most of the capacity to acquire thermotolerance, whereas the constitutive lines exhibited high levels of basal, uninduced resistance to elevated temperatures. Furthermore, the antisense plants produced seeds lacking both developmentally regulated
Hsp101 expression and the high level of basal thermotolerance characteristic of seeds and early germination. These changes in thermotolerance were the only detectable phenotypic consquences of the modulated Hsp101 levels. Hypocotyl elongation assays provided Hong and Vierling1 a powerful genetic screen for Arabidopsis mutants that have lost the capacity to acquire thermotolerance. Dramatically (and without bias towards particular gene loci), this approach yielded a mutant (hot1) with a Glu-to-Lys substitution in the second ATP-binding domain of Hsp101. These plants accumulated normal levels of inducible Hsp101 but failed to acquire thermotolerance. Perhaps, as suggested, the sessile lifestyle of plants requires the vigorous
capacity of Hsp101 to disassemble protein aggregates; animals, with their diverse thermoregulatory devices, make do with the refolding capacity of Hsp70 and members of other Hsp families. Characterization of the three other hot mutants is avidly awaited; crop protection applications are presumably under way.
1
2
Hong, S-W. and Vierling, E. (2000) Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc. Natl. Acad. Sci. U. S. A. 97, 4392–4397 Queitsch, C. et al. (2000) Heatshock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12, 479–492
How is the gate to the proteasome opened?
This month’s headlines were contributed by Donald Gullberg, Roy Golsteyn, Wallace Marshall, Robin May, Michael Mishkind and Cezary Wojcik.
312
For a long time, the 20S proteasome was conceived as an empty barrel with open ends, where substrates can get in. However, the crystallographic study of the yeast proteasome performed at a 2.4 Å resolution1 revealed that, unlike the archeal proteasome, the eukaryotic proteasome has sealed ends, and therefore a dilemma arose – how do the substrates enter the central proteolytic chamber? It was proposed that, when the proteasome binds to its activators, conformational changes occur that allow the entry of unfolded polypeptides, while small chromogenic
peptides might be able to access the central chamber by small side openings. Now, an elegant study by Osmulski and Gaczynska2, making use of the power of atomic force microscopy, has revealed that, to open the gates to the proteasome, there is no need for binding of additional proteins or activators. It seems that about 25% of yeast 20S proteasomes exist in an ‘open’ conformation, whereas 75% have sealed ends, in agreement with crystallographic data. However, the mere presence of standard proteasome substrates
is sufficient to shift the equilibrium towards ‘open’ proteasomes, which become the majority. Proteasome inhibitors do not increase the proportion of ‘closed’ proteasomes; however, they prevent the equilibrium shift induced by substrate binding. What impact will this finding have on proteasome research? Since the 20S proteasomes are able to open their gates even in the absence of activators, an old question resurfaces – are they able to perform a true role in the cell by themselves, without binding to activators and forming the 26S proteasome
trends in CELL BIOLOGY (Vol. 10) August 2000
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headlines complexes? It will also be interesting to discover how the substrate can drive such a huge conformational change as the opening of the proteasomal gates.
1
2
Groll, M. et al. (1997) Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386, 463–471 Osmulski, P.A. and Gaczynska, M.
(2000) Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast. J. Biol. Chem. 18, 13171–13174
Kendrin: a missing link between centrioles and spindle pole bodies The spindle pole bodies (SPBs) of yeast and fungi are the functional homologues of the centriole-based centrosomes found in larger eukaryotes such as humans. Phylogenetic evidence suggests that fungi (including yeast) and animals both evolved from a common ancestor species that contained centrioles, suggesting that the SPB might be a highly evolved form of centriole. However, despite great progress in recent years to identify the molecular components of SPBs, most known SPB proteins do not seem to have obvious homologues in animal cells. A recent report1 suggests that animal homologues of SPB proteins can indeed be found if one looks hard enough. One of the most central SPB structural proteins is SPC110, a calmodulin-binding protein. By
focusing on the calmodulin-binding region of SPC110, Flory and coworkers were able to identify a human gene encoding a similar calmodulinbinding site. This protein, called kendrin, had been identified previously and was known to be homologous to pericentrin, a centrosomal protein found in animal cells. Immunofluorescence indicated that kendrin does indeed localize to centrosomes, and high-resolution imaging suggested that it might be located in or near the centrioles themselves. The correspondence between kendrin and centrosomes was maintained in breast carcinoma cells, which frequently have multipolar spindles, and, in such cells, each spindle pole contained kendrin. Many questions about kendrin still remain. At present, there is no direct
evidence that kendrin function is required for centrosome functions, and the exact localization of kendrin relative to the centrioles remains to be determined. Nevertheless, these results provide encouragement that the detailed molecular studies on SPBs in yeast will ultimately be applicable to understanding the biology of centrosomes and centrioles in animal cells.
1
Flory, M.R. et al. (2000) Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. Proc. Natl. Acad. Sci. U. S. A. 97, 5919–5923
It’s not how old you are, but how old your T-cells feel For a biological phenomenon that affects all of us, the process of aging remains surprisingly poorly understood, mainly because of its intrinsic complexity. One of its most obvious features is a significant reduction in one’s ability to fight infection, yet the biological basis for this decline is far from clear. Now Sakata-Kaneko et al.1 have examined one facet of this process at a molecular level – and come up with a few surprises. The group isolated CD41 T cells (Th, or ‘helper’ T cells) from two groups of volunteers, one young and one old. These were then analysed for cytokine synthesis or expression of surface (CD) markers. The CD41 T cell population can be divided into two subsets, Th1 and Th2. Th1 produce cytokines such as interleukin 2 (IL-2) and interferon g (IFN-g) and play a central role in regulating cell-mediated immunity (CMI) against intracellular pathogens and tumours. Th2, on the other hand, produce IL-4, -5 and
-10 and are primarily concerned with humoral immunity to extracellular parasites. As elderly patients often show defective CMI, Sakata-Kaneko and colleagues predicted an age-related reduction in the Th1 population. In fact, their work clearly demonstrated the opposite. The older patients synthesized more IFN-g and had a higher proportion of Th1 cells than the younger group. Intriguingly, CD41 T cells from old patients showed increased IL-2 secretion (a Th1 response) before, as well as after, stimulation. The high basal (prestimulation) level of secretion is indicative of a large proportion of previously activated Th cells – so-called ‘memory cells’. Given the enhanced Th1 population in old subjects, why is it that the same patients often show a defect in CMI, a Th1 response? In a lucid discussion, the authors present several explanations. Foremost among them is the
trends in CELL BIOLOGY (Vol. 10) August 2000
concept that, although old patients have more Th1 cells, they might be unable to amplify individual antigenspecific cells in response to antigen exposure, leading to a broad but ineffective response. This might be amplified by a defective effector phase – other groups have already shown age-related deficiencies in natural killer and cytotoxic T-lymphocyte function. This reductionist approach to a complex problem has proven both informative and encouraging. Perhaps growing old won’t turn out to be such a complicated business after all.
1
Sakata-Kaneko, S. et al. (2000) Altered Th1/Th2 commitment in human CD41 T cells with ageing. Clin. Exp. Immunol. 120, 267–273
313
HEADLINES
TCB
30/6/00
8:58 am
Page 312
ANNOUNCEMENT Regular readers will no doubt be familiar with TCB Headlines which provide short, insightful summaries of the latest cell-biology research papers. But did you know that you can now access timely summaries of outstanding recently published papers on a daily basis through the Commentaries section of our recently enhanced BioMedNet online service? Authored by distinguished scientists, Commentaries provide a thought-provoking synopsis of scientific endeavour across the life sciences, including of course cell biology. Access to Commentaries is FREE. All you need do to find the Commentaries section of BioMedNet is go to http://www.bmn.com, click on the ‘News and Comment’ link and then press the Commentaries button (you might need to register as a BioMedNet member if you haven’t already). Selected commentaries may also appear subsequently as Headlines in your TCB magazine, and collectively Headlines will be archived electronically in the ‘Journal Club’ section of BioMedNet. As a further commitment to providing value for you our readers, we hope you appreciate the new look and feel of this month’s bigger TCB issue. As part of a programme of continuous investment in the Trends journals, which will unfold over the coming months, we will provide you more reviews, further cutting-edge analysis and thoughtful opinion articles; indeed – more essential coverage than ever before of your science. We welcome your comments and suggestions on these developments – contact the Editor at [email protected]
Hot plants, hot yeast Members of the heat-shock protein 70 (Hsp70) family act as central players when vertebrates and flies acquire tolerance to otherwise-lethal temperatures. Bacteria and yeast, by contrast, use the Hsp100 family in this role. The Vierling1,2 and Lindquist2 labs now place plants squarely in the microbial camp. In a direct assault on the problem, Queitch et al.2 generated transgenic Arabidopsis plants that underexpressed (by antisense and cosuppression methods) or constitutively accumulated Hsp101. Low-expressing plants lost most of the capacity to acquire thermotolerance, whereas the constitutive lines exhibited high levels of basal, uninduced resistance to elevated temperatures. Furthermore, the antisense plants produced seeds lacking both developmentally regulated
Hsp101 expression and the high level of basal thermotolerance characteristic of seeds and early germination. These changes in thermotolerance were the only detectable phenotypic consquences of the modulated Hsp101 levels. Hypocotyl elongation assays provided Hong and Vierling1 a powerful genetic screen for Arabidopsis mutants that have lost the capacity to acquire thermotolerance. Dramatically (and without bias towards particular gene loci), this approach yielded a mutant (hot1) with a Glu-to-Lys substitution in the second ATP-binding domain of Hsp101. These plants accumulated normal levels of inducible Hsp101 but failed to acquire thermotolerance. Perhaps, as suggested, the sessile lifestyle of plants requires the vigorous
capacity of Hsp101 to disassemble protein aggregates; animals, with their diverse thermoregulatory devices, make do with the refolding capacity of Hsp70 and members of other Hsp families. Characterization of the three other hot mutants is avidly awaited; crop protection applications are presumably under way.
1
2
Hong, S-W. and Vierling, E. (2000) Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc. Natl. Acad. Sci. U. S. A. 97, 4392–4397 Queitsch, C. et al. (2000) Heatshock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12, 479–492
How is the gate to the proteasome opened?
This month’s headlines were contributed by Donald Gullberg, Roy Golsteyn, Wallace Marshall, Robin May, Michael Mishkind and Cezary Wojcik.
312
For a long time, the 20S proteasome was conceived as an empty barrel with open ends, where substrates can get in. However, the crystallographic study of the yeast proteasome performed at a 2.4 Å resolution1 revealed that, unlike the archeal proteasome, the eukaryotic proteasome has sealed ends, and therefore a dilemma arose – how do the substrates enter the central proteolytic chamber? It was proposed that, when the proteasome binds to its activators, conformational changes occur that allow the entry of unfolded polypeptides, while small chromogenic
peptides might be able to access the central chamber by small side openings. Now, an elegant study by Osmulski and Gaczynska2, making use of the power of atomic force microscopy, has revealed that, to open the gates to the proteasome, there is no need for binding of additional proteins or activators. It seems that about 25% of yeast 20S proteasomes exist in an ‘open’ conformation, whereas 75% have sealed ends, in agreement with crystallographic data. However, the mere presence of standard proteasome substrates
is sufficient to shift the equilibrium towards ‘open’ proteasomes, which become the majority. Proteasome inhibitors do not increase the proportion of ‘closed’ proteasomes; however, they prevent the equilibrium shift induced by substrate binding. What impact will this finding have on proteasome research? Since the 20S proteasomes are able to open their gates even in the absence of activators, an old question resurfaces – are they able to perform a true role in the cell by themselves, without binding to activators and forming the 26S proteasome
trends in CELL BIOLOGY (Vol. 10) August 2000
TCB
08/00 paste-up
30/6/00
8:58 am
Page 313
headlines complexes? It will also be interesting to discover how the substrate can drive such a huge conformational change as the opening of the proteasomal gates.
1
2
Groll, M. et al. (1997) Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386, 463–471 Osmulski, P.A. and Gaczynska, M.
(2000) Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast. J. Biol. Chem. 18, 13171–13174
Kendrin: a missing link between centrioles and spindle pole bodies The spindle pole bodies (SPBs) of yeast and fungi are the functional homologues of the centriole-based centrosomes found in larger eukaryotes such as humans. Phylogenetic evidence suggests that fungi (including yeast) and animals both evolved from a common ancestor species that contained centrioles, suggesting that the SPB might be a highly evolved form of centriole. However, despite great progress in recent years to identify the molecular components of SPBs, most known SPB proteins do not seem to have obvious homologues in animal cells. A recent report1 suggests that animal homologues of SPB proteins can indeed be found if one looks hard enough. One of the most central SPB structural proteins is SPC110, a calmodulin-binding protein. By
focusing on the calmodulin-binding region of SPC110, Flory and coworkers were able to identify a human gene encoding a similar calmodulinbinding site. This protein, called kendrin, had been identified previously and was known to be homologous to pericentrin, a centrosomal protein found in animal cells. Immunofluorescence indicated that kendrin does indeed localize to centrosomes, and high-resolution imaging suggested that it might be located in or near the centrioles themselves. The correspondence between kendrin and centrosomes was maintained in breast carcinoma cells, which frequently have multipolar spindles, and, in such cells, each spindle pole contained kendrin. Many questions about kendrin still remain. At present, there is no direct
evidence that kendrin function is required for centrosome functions, and the exact localization of kendrin relative to the centrioles remains to be determined. Nevertheless, these results provide encouragement that the detailed molecular studies on SPBs in yeast will ultimately be applicable to understanding the biology of centrosomes and centrioles in animal cells.
1
Flory, M.R. et al. (2000) Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. Proc. Natl. Acad. Sci. U. S. A. 97, 5919–5923
It’s not how old you are, but how old your T-cells feel For a biological phenomenon that affects all of us, the process of aging remains surprisingly poorly understood, mainly because of its intrinsic complexity. One of its most obvious features is a significant reduction in one’s ability to fight infection, yet the biological basis for this decline is far from clear. Now Sakata-Kaneko et al.1 have examined one facet of this process at a molecular level – and come up with a few surprises. The group isolated CD41 T cells (Th, or ‘helper’ T cells) from two groups of volunteers, one young and one old. These were then analysed for cytokine synthesis or expression of surface (CD) markers. The CD41 T cell population can be divided into two subsets, Th1 and Th2. Th1 produce cytokines such as interleukin 2 (IL-2) and interferon g (IFN-g) and play a central role in regulating cell-mediated immunity (CMI) against intracellular pathogens and tumours. Th2, on the other hand, produce IL-4, -5 and
-10 and are primarily concerned with humoral immunity to extracellular parasites. As elderly patients often show defective CMI, Sakata-Kaneko and colleagues predicted an age-related reduction in the Th1 population. In fact, their work clearly demonstrated the opposite. The older patients synthesized more IFN-g and had a higher proportion of Th1 cells than the younger group. Intriguingly, CD41 T cells from old patients showed increased IL-2 secretion (a Th1 response) before, as well as after, stimulation. The high basal (prestimulation) level of secretion is indicative of a large proportion of previously activated Th cells – so-called ‘memory cells’. Given the enhanced Th1 population in old subjects, why is it that the same patients often show a defect in CMI, a Th1 response? In a lucid discussion, the authors present several explanations. Foremost among them is the
trends in CELL BIOLOGY (Vol. 10) August 2000
concept that, although old patients have more Th1 cells, they might be unable to amplify individual antigenspecific cells in response to antigen exposure, leading to a broad but ineffective response. This might be amplified by a defective effector phase – other groups have already shown age-related deficiencies in natural killer and cytotoxic T-lymphocyte function. This reductionist approach to a complex problem has proven both informative and encouraging. Perhaps growing old won’t turn out to be such a complicated business after all.
1
Sakata-Kaneko, S. et al. (2000) Altered Th1/Th2 commitment in human CD41 T cells with ageing. Clin. Exp. Immunol. 120, 267–273
313