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Clathrin – a new neurological target?
UPDATE
TARGETS Vol. 1, No. 6 December 2002
NEWS that hold the whole protein together. And in this case, the Trp residue is essential to that structure. ‘It snuggles in between the prolines,’ describes Roitberg;‘it forms a little sandwich.’ The protein is called Trp-cage, explains Andersen, because of the stabilizing structure made of proline residues that surround the Trp residue. ‘The burial of an indole ring is key to proper folding,’ which is achieved by the formation of hydrogen bonds. Andersen found with mutational studies that when another, similar residue (Phe or His) replaces Trp,‘the protein is totally unfolded,’ perhaps because of the inability of these residues to form H bonds [5]. Baker notes that the folding rate of a protein depends on its final topology – the conformation of these secondary structural elements – and that size usually does not
correlate well with folding rate [6]. However, in this case the protein is so small that it falls outside the usual size range, and correlations that hold true for other proteins could break down. ‘It’s no coincidence that the smallest is the fastest to fold,’ he adds. ‘It’s only 20 amino acids, so you know it won’t take half an hour,’ quips Hagen, but they were amazed at just how fast the protein assumed its final structural state.With a folding rate that is ‘bumping up against the physical limitations… it brings us closer to the idea of a diffusion-limited folding.’ Hagen asks, ‘Just how fast can a chain move?’ The researchers hope that Trp-cage will help answer that question, as well as others about general rules for folding in proteins of all sizes. ‘It’s going to be a fun, on-going thing,’ says Simmerling.
References 1 Qiu, L. et al. (2002) Smaller and faster: the 20-residue Trp-cage protein folds in 4 µs. J. Am. Chem. Soc. 124, 12952–12953 2 Neidigh, J.W. et al. (2002) Designing a 20-residue protein. Nature Struct. Bio. 9, 425–430 3 Cochran, A.G. et al. (2001) Tryptophan zippers: stable, monomeric β-hairpins. Proc. Natl. Acad. Sci. U. S. A. 98, 5578–5583. Erratum in: Proc. Natl. Acad. Sci. U. S. A. (2002) 99, 9081 4 Simmerling, et al. (2002) All-atom structure prediction and folding simulations of a stable protein. J. Am. Chem. Soc. 124, 11258–11259 5 Barua, B. and Andersen, N. (2002) Determinants of miniproteins: Can anything replace a buried H-bonded Trp side chain? Lett. Peptide Sci. 8, 221–226 6 Plaxco, K.W. et al. (2000) Topology, stability, sequence and length: defining the determinants of two-state protein folding kinetics. Biochemistry 39, 1177–1183
Clathrin – a new neurological target? Kathryn Senior, freelance writer
Recent research has revealed that endocytosis in postsynaptic neuronal membranes occurs at specialized ‘hot spots’ [1], rather than at random points all over the membrane as was previously thought. ‘There are defined points at which neurons take in drugs, receptors and other molecules from their surface membranes,’ comments Michael Ehlers from Duke University, North Carolina (http://www.duke.edu), who was senior author on the paper. Endocytosis is part of the constant cycling of receptors to and from the membrane surface. ‘This cycling process enables neurons to regulate the number of surface receptors for chemical triggers such as neurotransmitters and drugs, thereby controlling neuronal sensitivity to hormones, transmitters, and therapeutic agents,’ continues Ehlers.The newly discovered ‘endocytic zones’ are also entry points for nutrients and pathogens, such as viruses.
Searching out the elusive ‘hot spots’ Ehlers and colleagues investigated the localisation and dynamics of clathrin coats (a
184
www.drugdiscoverytoday.com
protein that coats membranes) in neuronal dendrites and neuronal spines, concentrating in particular on the postsynaptic region of the neuron.They used high-resolution fluorescent imaging of hippocampal neurons expressing clathrin–GFP (a fusion protein of clathrin light chain and the green fluorescent protein) to show that clathrin formation, clathrin coat dispersal and cargo transit into the cell are rapid, highly localized events. Intake of receptors tended to occur at specialized sites in the dendritic plasma membrane, notably in discrete areas at the tips of filopodia and in the lateral domains of mature neuronal spines [1].
Sites of clathrin assembly Gilbert Di Paolo, at Yale University School of Medicine (http://www.med.yale.edu) comments that this study is a new approach to investigating neuronal function. ‘Whereas most of the studies on clathrin-mediated endocytosis in neurons have so far focused on the presynaptic side and on the role of this process in synaptic vesicle recycling, the
authors in this study investigate the process in dendrites,’ he says. Previous seminal studies had reported the existence of endocytic ‘hot spots’ in non-neuronal cells [2] and Di Paolo points out that ‘one could have anticipated the existence of comparable dynamic structures in dendrites.’ However, he continues, ‘the originality in the authors’ study lies in the fact that the dynamics of these structures are developmentally-regulated.’ In young neurons, cycles of clathrin assembly and disassembly occur rapidly in dendrites, both in the shaft and at the tips of filopodia, whereas in mature neurons these cycles occur with much slower kinetics, reflecting the existence of more stable sites of endocytosis.
New insights into the cell biology of the neuron The endocytic hot spots lie laterally to the postsynaptic density – areas of the membrane that contain clusters of receptors – and occur independently of synaptic activity. ‘It follows therefore that synaptic receptors
1477-3627/02/$ – see front matter ©2002 Elsevier Science Ltd.All rights reserved. PII: S1477-3627(02)02272-9
UPDATE
TARGETS Vol. 1, No. 6 December 2002
NEWS IN BRIEF and other membrane proteins must be translocated to endocytic sites before internalisation, which spatially and temporally separates their functional inactivation from endocytosis,’ explains Ehlers.This observation is also important because it suggests that dendritic spines share architectural similarities with the presynaptic side. ‘In the presynaptic neuronal membrane, the areas where clathrin-mediated endocytosis occurs are spatially segregated from the exocytic zones. These areas, also called ‘periactive zones’, surround the active zones.Therefore, following fusion of synaptic vesicles with the plasmalemma, intrinsic synaptic vesicle proteins must diffuse laterally in the membrane to reach the periactive zones, from which synaptic vesicles can be recycled,’ explains Di Paolo.
Potential impact on drug development ‘As well as telling us more about the biology of the neuron, the discovery of ‘doorways’ into the postsynaptic membrane raises the possibility that drugs affecting receptor transport to and through the zones could prove useful in treating addiction, depression,
stroke, epilepsy and other neurological disorders that involve abnormal transport of receptors into the neuron,’ suggests Ehlers. For example, tolerance to drugs of abuse, a frequent feature of addiction, as well as the lost responsiveness to certain anti-depressants, anti-psychotics and anti-anxiety agent over time, involves removal of surface receptors.‘This removal probably occurs by transport through the endocytic zones we have identified,’ says Ehlers.The opposite situation is also true: ineffective removal of surface receptors and ion channels could lead to vulnerability of neurons to damage and dysfunction during stroke and epileptic states.
Clathrin and drug discovery Di Paolo points out that clathrin dynamics are dependent on a dynamic actin cytoskeleton and that drugs that are active against actin dramatically affect clathrin dynamics, both in young and mature neurons.There is a growing list of studies showing a tight link between clathrin-mediated endocytosis and the actin cytoskeleton. However, the way actin assists endocytosis is still poorly understood. Di Paolo warns that,‘in terms of drug discovery, it is probably too premature to think
News in brief Targets and mechanisms HSV-2 implicated in cervical cancer Herpes simplex virus-2 (HSV-2) could be an accomplice of human papillomavirus (HPV) in some cases of cervical cancer [1].This new finding could lead to a greater understanding of how cervical cancer develops and how it might be prevented. Infection with HPV is known to be the main cause of invasive cervical cancer. However, factors such as smoking, number of live births
and contraceptive use are known to act in conjunction with HPV to increase the risk of the disease. Now, it seems that infection with HSV-2 can also increase susceptibility. Researchers led by Jennifer Smith, of the International Agency for Research on Cancer (http://www.iarc.fr/), analyzed samples from 1263 women with invasive cervical cancer and 1117 women without the disease.They found antibodies to HSV-2 (an indicator of past HSV-2 infection) in the blood of >40% of women with invasive cervical cancer but in only 25.6% of control women. Affected patients whose cervical samples contained HPV DNA were at least twice as likely to develop cervical carcinoma if they had also been infected with HSV-2. The authors stress that the effect of HSV-2 on susceptibility to cervical neoplasia is modest
1477-3627/02/$ – see front matter ©2002 Elsevier Science Ltd.All rights reserved. PII: S1477-3627(02)02275-4
of targeting clathrin-mediated endocytosis of postsynaptic receptors with the hope of affecting synaptic plasticity,’ Much effort must be devoted to elucidating the molecular mechanisms underlying their internalization first, he says. Nevertheless, Ehlers is optimistic: ‘Our current findings raise the possibility of developing entirely new categories of drugs that target the movement of receptors and channels from one site in the surface membrane to the endocytic zone, or that modulate the endocytic zone itself,’ predicts Ehlers. These possibilities are still quite theoretical, says Ehlers, but they provide a glimpse of the kinds of approaches and new classes of drug targets that could be developed in the future. ‘We now have the tools and the cell biological knowledge to start testing this idea, although much work remains to be done’, he concludes.
References 1 Blanpied, T.A. et al. (2002) Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron 36, 435–449 2 Gaidarov, I. et al. (1999) Spatial control of coated-pit dynamics in living cells. Nat. Cell. Biol. 1, 1–7
compared with that of HPV. However, they suggest that:‘Further studies of the natural history of HPV in relation with other cervical pathogens may provide insights into the mechanisms by which ulcerative sexually transmitted infections and inflammation contribute to the development of the malignant phenotype and may suggest novel approaches to the prevention of cervical neoplasia.’ 1 Smith, J.S. et al. (2002) Herpes simplex virus-2 as a human papillomavirus cofactor in the etiology of invasive cervical cancer. J. Natl. Cancer Inst. 94, 1604–1613
Virus linked to colon cancer A virus endemic to the human population might be a cause of cancer, according to the latest scientific research [2]. Human cytomegalovirus (CMV) is a β-herpes virus, which usually goes unnoticed in healthy people, but can be life-threatening in patients with weakened immune systems.
www.drugdiscoverytoday.com
185
TARGETS Vol. 1, No. 6 December 2002
NEWS that hold the whole protein together. And in this case, the Trp residue is essential to that structure. ‘It snuggles in between the prolines,’ describes Roitberg;‘it forms a little sandwich.’ The protein is called Trp-cage, explains Andersen, because of the stabilizing structure made of proline residues that surround the Trp residue. ‘The burial of an indole ring is key to proper folding,’ which is achieved by the formation of hydrogen bonds. Andersen found with mutational studies that when another, similar residue (Phe or His) replaces Trp,‘the protein is totally unfolded,’ perhaps because of the inability of these residues to form H bonds [5]. Baker notes that the folding rate of a protein depends on its final topology – the conformation of these secondary structural elements – and that size usually does not
correlate well with folding rate [6]. However, in this case the protein is so small that it falls outside the usual size range, and correlations that hold true for other proteins could break down. ‘It’s no coincidence that the smallest is the fastest to fold,’ he adds. ‘It’s only 20 amino acids, so you know it won’t take half an hour,’ quips Hagen, but they were amazed at just how fast the protein assumed its final structural state.With a folding rate that is ‘bumping up against the physical limitations… it brings us closer to the idea of a diffusion-limited folding.’ Hagen asks, ‘Just how fast can a chain move?’ The researchers hope that Trp-cage will help answer that question, as well as others about general rules for folding in proteins of all sizes. ‘It’s going to be a fun, on-going thing,’ says Simmerling.
References 1 Qiu, L. et al. (2002) Smaller and faster: the 20-residue Trp-cage protein folds in 4 µs. J. Am. Chem. Soc. 124, 12952–12953 2 Neidigh, J.W. et al. (2002) Designing a 20-residue protein. Nature Struct. Bio. 9, 425–430 3 Cochran, A.G. et al. (2001) Tryptophan zippers: stable, monomeric β-hairpins. Proc. Natl. Acad. Sci. U. S. A. 98, 5578–5583. Erratum in: Proc. Natl. Acad. Sci. U. S. A. (2002) 99, 9081 4 Simmerling, et al. (2002) All-atom structure prediction and folding simulations of a stable protein. J. Am. Chem. Soc. 124, 11258–11259 5 Barua, B. and Andersen, N. (2002) Determinants of miniproteins: Can anything replace a buried H-bonded Trp side chain? Lett. Peptide Sci. 8, 221–226 6 Plaxco, K.W. et al. (2000) Topology, stability, sequence and length: defining the determinants of two-state protein folding kinetics. Biochemistry 39, 1177–1183
Clathrin – a new neurological target? Kathryn Senior, freelance writer
Recent research has revealed that endocytosis in postsynaptic neuronal membranes occurs at specialized ‘hot spots’ [1], rather than at random points all over the membrane as was previously thought. ‘There are defined points at which neurons take in drugs, receptors and other molecules from their surface membranes,’ comments Michael Ehlers from Duke University, North Carolina (http://www.duke.edu), who was senior author on the paper. Endocytosis is part of the constant cycling of receptors to and from the membrane surface. ‘This cycling process enables neurons to regulate the number of surface receptors for chemical triggers such as neurotransmitters and drugs, thereby controlling neuronal sensitivity to hormones, transmitters, and therapeutic agents,’ continues Ehlers.The newly discovered ‘endocytic zones’ are also entry points for nutrients and pathogens, such as viruses.
Searching out the elusive ‘hot spots’ Ehlers and colleagues investigated the localisation and dynamics of clathrin coats (a
184
www.drugdiscoverytoday.com
protein that coats membranes) in neuronal dendrites and neuronal spines, concentrating in particular on the postsynaptic region of the neuron.They used high-resolution fluorescent imaging of hippocampal neurons expressing clathrin–GFP (a fusion protein of clathrin light chain and the green fluorescent protein) to show that clathrin formation, clathrin coat dispersal and cargo transit into the cell are rapid, highly localized events. Intake of receptors tended to occur at specialized sites in the dendritic plasma membrane, notably in discrete areas at the tips of filopodia and in the lateral domains of mature neuronal spines [1].
Sites of clathrin assembly Gilbert Di Paolo, at Yale University School of Medicine (http://www.med.yale.edu) comments that this study is a new approach to investigating neuronal function. ‘Whereas most of the studies on clathrin-mediated endocytosis in neurons have so far focused on the presynaptic side and on the role of this process in synaptic vesicle recycling, the
authors in this study investigate the process in dendrites,’ he says. Previous seminal studies had reported the existence of endocytic ‘hot spots’ in non-neuronal cells [2] and Di Paolo points out that ‘one could have anticipated the existence of comparable dynamic structures in dendrites.’ However, he continues, ‘the originality in the authors’ study lies in the fact that the dynamics of these structures are developmentally-regulated.’ In young neurons, cycles of clathrin assembly and disassembly occur rapidly in dendrites, both in the shaft and at the tips of filopodia, whereas in mature neurons these cycles occur with much slower kinetics, reflecting the existence of more stable sites of endocytosis.
New insights into the cell biology of the neuron The endocytic hot spots lie laterally to the postsynaptic density – areas of the membrane that contain clusters of receptors – and occur independently of synaptic activity. ‘It follows therefore that synaptic receptors
1477-3627/02/$ – see front matter ©2002 Elsevier Science Ltd.All rights reserved. PII: S1477-3627(02)02272-9
UPDATE
TARGETS Vol. 1, No. 6 December 2002
NEWS IN BRIEF and other membrane proteins must be translocated to endocytic sites before internalisation, which spatially and temporally separates their functional inactivation from endocytosis,’ explains Ehlers.This observation is also important because it suggests that dendritic spines share architectural similarities with the presynaptic side. ‘In the presynaptic neuronal membrane, the areas where clathrin-mediated endocytosis occurs are spatially segregated from the exocytic zones. These areas, also called ‘periactive zones’, surround the active zones.Therefore, following fusion of synaptic vesicles with the plasmalemma, intrinsic synaptic vesicle proteins must diffuse laterally in the membrane to reach the periactive zones, from which synaptic vesicles can be recycled,’ explains Di Paolo.
Potential impact on drug development ‘As well as telling us more about the biology of the neuron, the discovery of ‘doorways’ into the postsynaptic membrane raises the possibility that drugs affecting receptor transport to and through the zones could prove useful in treating addiction, depression,
stroke, epilepsy and other neurological disorders that involve abnormal transport of receptors into the neuron,’ suggests Ehlers. For example, tolerance to drugs of abuse, a frequent feature of addiction, as well as the lost responsiveness to certain anti-depressants, anti-psychotics and anti-anxiety agent over time, involves removal of surface receptors.‘This removal probably occurs by transport through the endocytic zones we have identified,’ says Ehlers.The opposite situation is also true: ineffective removal of surface receptors and ion channels could lead to vulnerability of neurons to damage and dysfunction during stroke and epileptic states.
Clathrin and drug discovery Di Paolo points out that clathrin dynamics are dependent on a dynamic actin cytoskeleton and that drugs that are active against actin dramatically affect clathrin dynamics, both in young and mature neurons.There is a growing list of studies showing a tight link between clathrin-mediated endocytosis and the actin cytoskeleton. However, the way actin assists endocytosis is still poorly understood. Di Paolo warns that,‘in terms of drug discovery, it is probably too premature to think
News in brief Targets and mechanisms HSV-2 implicated in cervical cancer Herpes simplex virus-2 (HSV-2) could be an accomplice of human papillomavirus (HPV) in some cases of cervical cancer [1].This new finding could lead to a greater understanding of how cervical cancer develops and how it might be prevented. Infection with HPV is known to be the main cause of invasive cervical cancer. However, factors such as smoking, number of live births
and contraceptive use are known to act in conjunction with HPV to increase the risk of the disease. Now, it seems that infection with HSV-2 can also increase susceptibility. Researchers led by Jennifer Smith, of the International Agency for Research on Cancer (http://www.iarc.fr/), analyzed samples from 1263 women with invasive cervical cancer and 1117 women without the disease.They found antibodies to HSV-2 (an indicator of past HSV-2 infection) in the blood of >40% of women with invasive cervical cancer but in only 25.6% of control women. Affected patients whose cervical samples contained HPV DNA were at least twice as likely to develop cervical carcinoma if they had also been infected with HSV-2. The authors stress that the effect of HSV-2 on susceptibility to cervical neoplasia is modest
1477-3627/02/$ – see front matter ©2002 Elsevier Science Ltd.All rights reserved. PII: S1477-3627(02)02275-4
of targeting clathrin-mediated endocytosis of postsynaptic receptors with the hope of affecting synaptic plasticity,’ Much effort must be devoted to elucidating the molecular mechanisms underlying their internalization first, he says. Nevertheless, Ehlers is optimistic: ‘Our current findings raise the possibility of developing entirely new categories of drugs that target the movement of receptors and channels from one site in the surface membrane to the endocytic zone, or that modulate the endocytic zone itself,’ predicts Ehlers. These possibilities are still quite theoretical, says Ehlers, but they provide a glimpse of the kinds of approaches and new classes of drug targets that could be developed in the future. ‘We now have the tools and the cell biological knowledge to start testing this idea, although much work remains to be done’, he concludes.
References 1 Blanpied, T.A. et al. (2002) Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron 36, 435–449 2 Gaidarov, I. et al. (1999) Spatial control of coated-pit dynamics in living cells. Nat. Cell. Biol. 1, 1–7
compared with that of HPV. However, they suggest that:‘Further studies of the natural history of HPV in relation with other cervical pathogens may provide insights into the mechanisms by which ulcerative sexually transmitted infections and inflammation contribute to the development of the malignant phenotype and may suggest novel approaches to the prevention of cervical neoplasia.’ 1 Smith, J.S. et al. (2002) Herpes simplex virus-2 as a human papillomavirus cofactor in the etiology of invasive cervical cancer. J. Natl. Cancer Inst. 94, 1604–1613
Virus linked to colon cancer A virus endemic to the human population might be a cause of cancer, according to the latest scientific research [2]. Human cytomegalovirus (CMV) is a β-herpes virus, which usually goes unnoticed in healthy people, but can be life-threatening in patients with weakened immune systems.
www.drugdiscoverytoday.com
185