- Email: [email protected]
Hope for a broken heart?
Update
TRENDS in Biotechnology
Vol.22 No.10 October 2004
Research Focus
Hope for a broken heart? Patrick Most, Carmen Eicher, Mirko Vo¨lkers, Sven T. Pleger and Hugo A. Katus Division of Cardiology, Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
Heart failure affects 23 million people worldwide and results from cardiac dysfunction characterized by decreased responsiveness to b-adrenergic stimulation. A recent publication by W.J. Koch and colleagues highlights evidence for targeted b-adrenergic receptor kinase (bARK1) inhibition by gene transfer to improve contractile function and b-adrenergic responsiveness in failing human myocardium. This proof-of-concept study has great importance for future heart failure therapy because it provides evidence for the therapeutic effectiveness of bARK1 inhibition in failing human myocardium. Approximately 23 million people worldwide are afflicted with congestive heart failure (HF), and 2 million new cases of the disease are diagnosed each year worldwide. In contrast to other cardiovascular disorders that have declined during past decades, the incidence of HF is on the rise. Although advances in pharmacology have led to better treatment, 50% of patients with the most advanced stage of HF die within a year. This sobering statistic highlights the need for novel therapeutic approaches for treating this progressive disease. However, our understanding of HF on a molecular level has progressed, and therapies that target the deranged molecular signaling characteristic of the disease hold tremendous promise for the future. In this regard, a recent publication of Koch and coworkers has provided evidence for the inhibition of elevated b-adrenergic receptor kinase 1 (bARK1) activity in failing human myocardium by adenoviral gene transfer of a peptide inhibitor of bARK1 (bARKct) to improve contractile function and b-adrenergic responsiveness [1]. This finding has tremendous therapeutic implications because bARK1 inhibition either by cardiac bARKct gene transfer or more directly by the development of suitable inhibitor drugs represents a novel therapeutic means for the clinical treatment of human heart failure. Role of bARK1 in normal heart function: manipulating the power switch Under normal physiological conditions, the classic stressor exercise-related ‘flight or fight’ response involves acute activation of the sympathetic nervous system. The subsequent catecholamine release in turn immediately augments cardiac output and systemic blood pressure by increasing heart rate and enhancing contractility. These Corresponding author: Patrick Most ([email protected]). Available online 28 August 2004 www.sciencedirect.com
effects are the result of enhanced b-adrenergic receptor (AR) signaling in the heart. Activity of these G proteincoupled receptors (GPCRs) is tightly regulated through a complex network of interdependent factors that allow acute catecholamine stimulation but apparently prevent chronic b-AR activation that is toxic to the heart [2]. In this regard, non-sustained activation of GPCR kinases (GRKs) provides an important intrinsic mechanism for turning off agonist-occupied GPCR signaling through receptor phosphorylation. This results both in desensitization – rapid uncoupling of the receptor from its effector molecules (e.g. G proteins) and in downregulation – delayed net loss of receptors from the cell surface (e.g. by internalization and degradation) [3]. In the heart, the b-AR kinase (bARK1), also known as GRK2, is the primarily expressed GRK isoform, which desensitizes b-AR and other GPCRs via membrane translocation dependent on direct binding to dissociated bg-subunits of G proteins (Gbg) [4]. Using different genetic strategies to manipulate the concentration and activity of bARK1 in the hearts of mice, Koch and colleagues have shown in a recent series of elegant experiments that in the normal heart, the level of contractile function and the heart’s response to catecholamine stimulation is essentially determined by bARK1 activity [5–7]. Cardiacrestricted overexpression of the full-length protein decreased cardiac contractility and b-AR responsiveness [5]. By contrast, overexpression of a peptide inhibitor of bARK1, made up of the last 194 amino acids of the receptor kinase (bARKct) and ablating the bARK1 gene caused heightened contractile strength and response to b-AR stimuli [5,6]. Hence, this physiological reciprocation demonstrates a crucial role for bARK1 in normal cardiac regulation and function.
bARK1 function in the failing heart: the dark side of the kinase Understanding bARK1 function in cardiovascular physiology has important consequences for cardiac disease states that are characterized by increased bARK1 activity and expression, such as myocardial hypertrophy and HF [8], because even partial inhibition of bARK1 activity could lead to improved functional responsiveness to catecholamines. As the heart begins to fail, compensatory mechanisms involving chronic stimulation of the sympathetic nervous system are initiated to maintain cardiac output and systemic blood pressure [3]. However, the ongoing hyperadrenergic state triggers a sequence of maladaptive events, including b-AR desensitization and
488
Update
TRENDS in Biotechnology
downregulation, which in turn lead to impaired contractility and loss of b-AR-dependent contractile reserve [4]. This effect is mediated at least in part both by concurrent elevation in the expression and sustained activation of bARK1, which is considered to be among the earliest changes noted in heart disease. However, this not only results in the attenuation of b-AR signaling, but is also associated with altered coupling of GPCRs to other signaling pathways that are involved in ventricular remodelling, such as the mitogen-activated protein (MAP) kinase, phosphatidyl-inositol-3 kinase (PtdIns(3)K) and the calcineurin cascades, driving the progressive downhill clinical course of patients with HF [2]. However, Koch and colleagues have previously shown that bARK1 inhibition by cardiac-restricted transgenic overexpression of bARKct can rescue several murine models of severe HF, imparting improvement in myocardial b-AR signaling, left ventricular function and remodeling, cardiac hypertrophy and survival [9–11]. Thus, their results strongly support the view that sustained bARK1 activity in the failing heart is rather maladaptive than protective and inhibition of elevated bARK1 activity in HF could represent a novel approach for treating the disease. bARK1 inhibition in HF: therapeutic implications In their current study [1], Koch and colleagues successfully tested this therapeutic strategy for the first time in failing human myocardium. Taking advantage of adenoviral-mediated bARKct overexpression, they demonstrated that inhibition of enhanced bARK1 activity in human failing ventricular cardiomyocytes improves contractile function and b-AR responsiveness. Although this study is limited by the absence of non-failing cardiomyocytes, these results provide a ‘proof-of-principle’ for the therapeutic effectiveness of bARK1 inhibition in failing human myocardium. Additional studies by this group have already tested these beneficial effects in larger animal HF models by intracoronary delivery of replication-deficient adenoviruses encoding the bARKct transgene to achieve global cardiac gene delivery [12] – a prerequisite for application of gene therapy in human heart failure. These encouraging results provide a strong argument to move into first clinical trials. Already gene therapy for cardiac ischemia has reached human clinical trials, and early results of phase I clinical trials that employed adenoviral vectors to deliver either vascular endothelial growth factor (Ad-VEGF) or fibroblast growth factor 4 (Ad-FGF-4) to the ischemic heart showed no adverse events that could be attributed to adenovirus administration [12]. In this regard, bARKct gene therapy of human HF awaits application in the clinical arena. However, a major concern about bARK1 inhibition still stems from the notion that treatment of HF through enhancement, rather that supression of b-AR signaling, runs counter to current conventional wisdom in the clinical therapy of HF. This notion is based on the observation that application of b-AR agonists has been shown to be detrimental, whereas b-AR blockade has remarkable salutary effects in HF. It therefore seems important to note that b-blockade by carvedilol decreases the expression and activity of bARK1 in the hearts of mice www.sciencedirect.com
Vol.22 No.10 October 2004
chronically treated with this agent [13]. Thus, decreased bARK1 and resultant enhancement of cardiac b-AR signaling might represent, at least in part, a possible mechanism for the positive beneficial effect seen by b-AR blockade in HF. Moreover, Koch and colleagues have already demonstrated that cardiac bARK1 inhibition by bARKct, together with b-AR blockade by metoprolol, act synergistically to reduce lethal arrhythmias and promote favourable remodelling in HF [10]. This has been shown in a murine model of severe HF in which cardiac bARK1 inhibition prolongs survival and potentiated b-AR antagonist therapy [10] – an interesting finding given recent clinical results that show improved survival of patients with chronic HF that were treated with b-blockers. Therefore, these interventions appear to function synergistically rather than opposing each other and, so far, bARK1 inhibition cannot be explained by any existing pharmacological paradigm. However, it should be remembered that bARKct also functions more generally to sequester Gbg–subunits from other biologically important GPCRs (e.g. angiotensin II and endothelin receptors) that are involved in the activation of hypertrophic pathways, and there might be inhibition of other as yet unknown Gbg–effectors [3]. The extent (if any) to which interruption of such interactions might contribute to the salutary effects of bARKct remains to be determined. bARK1 inhibition in HF: a clinical perspective The current results provided by Koch and coworkers [1] clearly demonstrate a novel therapeutic principle in human failing myocardium that awaits extension to clinical practice. To achieve this objective, it will be necessary to make further advances in gene-delivery technology and vector design to effectively test this option in the clinical arena. Current gene-delivery methods remain quite invasive. Engineered vectors with tissue tropism (e.g. AAV) that obviate the need for direct myocardial delivery would represent a significant advance. Furthermore, research has not yielded any method to control expression after gene delivery. Analogous to a physician adjusting a dose to produce a desired effect, finer exogenous control of transgene expression would be desirable. Moreover, in addition to bARKct gene therapy, recent advances in the field of drug-development might facilitate the development of small-molecule inhibitors of bARK1 and its interaction with Gbg that might add new drugs to the therapeutic armamentarium for the treatment of heart failure. References 1 Williams, M.L. et al. (2004) Targeted b-adrenergic receptor kinase (bARK1) inhibition by gene transfer in failing human hearts. Circulation 109, 1590–1593 2 Rockman, H.A. et al. (2002) Seven-transmembrane-spanning receptors and heart function. Nature 415, 206–212 3 Koch, W.J. et al. (2000) Functional consequences of altering myocardial adrenergic receptor signaling. Annu. Rev. Physiol. 62, 237–260 4 Lefkowitz, R.J. et al. (2000) Catecholamines, cardiac beta-adrenergic receptors, and heart failure. Circulation 101, 1634–1637 5 Koch, W.J. et al. (1995) Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science 268, 1350–1353
Update
TRENDS in Biotechnology
6 Jaber, M. et al. (1996) Essential role of b-adrenergic receptor kinase-1 in cardiac development and function. Proc. Natl. Acad. Sci. U. S. A. 93, 12974–12979 7 Rockman, H.A. et al. (1998) Control of myocardial contractile function by the level of b-adrenergic receptor kinase-1 in gene-targeted mice. J. Biol. Chem. 273, 18180–18184 8 Ungerer, M. et al. (1993) Altered expression of b-adrenergic receptor kinase and b1-adrenergic receptors in the failing heart. Circulation 87, 454–463 9 Rockman, H.A. et al. (1998) Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc. Natl. Acad. Sci. U. S. A. 95, 7000–7005 10 Harding, V.B. et al. (2001) Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure. Proc. Natl. Acad. Sci. U. S. A. 98, 5809–5814
Vol.22 No.10 October 2004
489
11 Freeman, K. et al. (2001) Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J. Clin. Invest. 107, 967–974 12 Williams, M.L. and Koch, W.J. (2004) Viral-based myocardial gene therapy approaches to alter cardiac function. Annu. Rev. Physiol. 66, 49–75 13 Iaccarino, G. et al. (1998) Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by b-adrenergic receptor stimulation and blockade. Circulation 98, 1783–1789
0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.08.004
Tailor-made glycoproteins Linda C. Hsieh–Wilson Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
Posttranslational modifications are a fundamental mechanism for the regulation of cellular physiology and function. A recent paper by Zhang et al. provides a novel strategy for the generation of homogeneous glycoproteins. The ability to install covalent modifications site-specifically into proteins holds tremendous promise for deciphering the role of posttranslational modifications and has exciting implications for the development of protein therapeutics. Given that the complexity of higher organisms is encoded in a remarkably small number of genes, attention has focused on other mechanisms to account for the diversity of biological systems. Protein posttranslational modifications (PTMs) represent a major source of variation – the covalent attachment of phosphate, lipid, and other chemical groups to amino acid side chains extends the capabilities of proteins and provides selective and temporal control over protein function. At a cellular level, PTMs have been shown to regulate vital processes such as transcription, cell-cycle progression, and programmed cell death. Protein glycosylation, the most abundant and complex form of PTM, requires an estimated 1% of mammalian genes [1] and distinguishes eukaryotic cells from simpler prokaryotic systems. Recent studies are challenging early views of carbohydrates as non-specific, static structures and are revealing exciting physiological roles for glycosylation. For example, Wei et al. have shown that HIV evades the immune system by evolving a dynamically changing shield of carbohydrates [2]. Glycosaminoglycans, another class of carbohydrates, play crucial roles during development, with genetic disruption of Corresponding author: Linda C. Hsieh–Wilson ([email protected]). Available online 28 August 2004 www.sciencedirect.com
glycosaminoglycan biosynthesis associated with severe growth defects [3]. The complex sulfation patterns present in glycosaminoglycans, which are tissue-specific, tightly regulated, and crucial for growth factor activation, suggest an intricate relationship between carbohydrate structure and biological function [3]. In the intracellular context, dynamic forms of glycosylation have been identified, such as the addition of b-N-acetylglucosamine to serine or threonine residues of proteins (O-GlcNAc glycosylation) [4]. Recent studies have linked O-GlcNAc glycosylation to the regulation of transcription factors such as cAMP-responsive element binding protein (CREB) [5] and the proteasome [6], raising the exciting possibility that this carbohydrate modification plays a fundamental role in cellular communication and protein function. Despite many important contributions, the diverse roles of glycosylation and other covalent modifications are only beginning to be understood. Detailed studies of their biological effects have been hindered by the dynamic nature and complexity of PTMs in vivo. For instance, the presence of a given modification can depend on the cell type or status (e.g. stimulated vs non-stimulated, normal vs diseased). It can also be restricted to subcellular compartments or protein subpopulations and can occur in combination with other PTMs. Except for protein phosphorylation, in which an aspartic or glutamic acid can sometimes mimic the phosphorylated residue, no natural amino acid can serve as an effective surrogate for glycosylation and other PTMs. Moreover, efforts to purify modified proteins for biochemical studies generally require abundant sources and extensive purification. Even when available, the purified proteins are frequently heterogeneous, bearing incomplete or multiple modifications. A recent paper by Schultz and colleagues provides a novel solution to these problems and should considerably
TRENDS in Biotechnology
Vol.22 No.10 October 2004
Research Focus
Hope for a broken heart? Patrick Most, Carmen Eicher, Mirko Vo¨lkers, Sven T. Pleger and Hugo A. Katus Division of Cardiology, Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
Heart failure affects 23 million people worldwide and results from cardiac dysfunction characterized by decreased responsiveness to b-adrenergic stimulation. A recent publication by W.J. Koch and colleagues highlights evidence for targeted b-adrenergic receptor kinase (bARK1) inhibition by gene transfer to improve contractile function and b-adrenergic responsiveness in failing human myocardium. This proof-of-concept study has great importance for future heart failure therapy because it provides evidence for the therapeutic effectiveness of bARK1 inhibition in failing human myocardium. Approximately 23 million people worldwide are afflicted with congestive heart failure (HF), and 2 million new cases of the disease are diagnosed each year worldwide. In contrast to other cardiovascular disorders that have declined during past decades, the incidence of HF is on the rise. Although advances in pharmacology have led to better treatment, 50% of patients with the most advanced stage of HF die within a year. This sobering statistic highlights the need for novel therapeutic approaches for treating this progressive disease. However, our understanding of HF on a molecular level has progressed, and therapies that target the deranged molecular signaling characteristic of the disease hold tremendous promise for the future. In this regard, a recent publication of Koch and coworkers has provided evidence for the inhibition of elevated b-adrenergic receptor kinase 1 (bARK1) activity in failing human myocardium by adenoviral gene transfer of a peptide inhibitor of bARK1 (bARKct) to improve contractile function and b-adrenergic responsiveness [1]. This finding has tremendous therapeutic implications because bARK1 inhibition either by cardiac bARKct gene transfer or more directly by the development of suitable inhibitor drugs represents a novel therapeutic means for the clinical treatment of human heart failure. Role of bARK1 in normal heart function: manipulating the power switch Under normal physiological conditions, the classic stressor exercise-related ‘flight or fight’ response involves acute activation of the sympathetic nervous system. The subsequent catecholamine release in turn immediately augments cardiac output and systemic blood pressure by increasing heart rate and enhancing contractility. These Corresponding author: Patrick Most ([email protected]). Available online 28 August 2004 www.sciencedirect.com
effects are the result of enhanced b-adrenergic receptor (AR) signaling in the heart. Activity of these G proteincoupled receptors (GPCRs) is tightly regulated through a complex network of interdependent factors that allow acute catecholamine stimulation but apparently prevent chronic b-AR activation that is toxic to the heart [2]. In this regard, non-sustained activation of GPCR kinases (GRKs) provides an important intrinsic mechanism for turning off agonist-occupied GPCR signaling through receptor phosphorylation. This results both in desensitization – rapid uncoupling of the receptor from its effector molecules (e.g. G proteins) and in downregulation – delayed net loss of receptors from the cell surface (e.g. by internalization and degradation) [3]. In the heart, the b-AR kinase (bARK1), also known as GRK2, is the primarily expressed GRK isoform, which desensitizes b-AR and other GPCRs via membrane translocation dependent on direct binding to dissociated bg-subunits of G proteins (Gbg) [4]. Using different genetic strategies to manipulate the concentration and activity of bARK1 in the hearts of mice, Koch and colleagues have shown in a recent series of elegant experiments that in the normal heart, the level of contractile function and the heart’s response to catecholamine stimulation is essentially determined by bARK1 activity [5–7]. Cardiacrestricted overexpression of the full-length protein decreased cardiac contractility and b-AR responsiveness [5]. By contrast, overexpression of a peptide inhibitor of bARK1, made up of the last 194 amino acids of the receptor kinase (bARKct) and ablating the bARK1 gene caused heightened contractile strength and response to b-AR stimuli [5,6]. Hence, this physiological reciprocation demonstrates a crucial role for bARK1 in normal cardiac regulation and function.
bARK1 function in the failing heart: the dark side of the kinase Understanding bARK1 function in cardiovascular physiology has important consequences for cardiac disease states that are characterized by increased bARK1 activity and expression, such as myocardial hypertrophy and HF [8], because even partial inhibition of bARK1 activity could lead to improved functional responsiveness to catecholamines. As the heart begins to fail, compensatory mechanisms involving chronic stimulation of the sympathetic nervous system are initiated to maintain cardiac output and systemic blood pressure [3]. However, the ongoing hyperadrenergic state triggers a sequence of maladaptive events, including b-AR desensitization and
488
Update
TRENDS in Biotechnology
downregulation, which in turn lead to impaired contractility and loss of b-AR-dependent contractile reserve [4]. This effect is mediated at least in part both by concurrent elevation in the expression and sustained activation of bARK1, which is considered to be among the earliest changes noted in heart disease. However, this not only results in the attenuation of b-AR signaling, but is also associated with altered coupling of GPCRs to other signaling pathways that are involved in ventricular remodelling, such as the mitogen-activated protein (MAP) kinase, phosphatidyl-inositol-3 kinase (PtdIns(3)K) and the calcineurin cascades, driving the progressive downhill clinical course of patients with HF [2]. However, Koch and colleagues have previously shown that bARK1 inhibition by cardiac-restricted transgenic overexpression of bARKct can rescue several murine models of severe HF, imparting improvement in myocardial b-AR signaling, left ventricular function and remodeling, cardiac hypertrophy and survival [9–11]. Thus, their results strongly support the view that sustained bARK1 activity in the failing heart is rather maladaptive than protective and inhibition of elevated bARK1 activity in HF could represent a novel approach for treating the disease. bARK1 inhibition in HF: therapeutic implications In their current study [1], Koch and colleagues successfully tested this therapeutic strategy for the first time in failing human myocardium. Taking advantage of adenoviral-mediated bARKct overexpression, they demonstrated that inhibition of enhanced bARK1 activity in human failing ventricular cardiomyocytes improves contractile function and b-AR responsiveness. Although this study is limited by the absence of non-failing cardiomyocytes, these results provide a ‘proof-of-principle’ for the therapeutic effectiveness of bARK1 inhibition in failing human myocardium. Additional studies by this group have already tested these beneficial effects in larger animal HF models by intracoronary delivery of replication-deficient adenoviruses encoding the bARKct transgene to achieve global cardiac gene delivery [12] – a prerequisite for application of gene therapy in human heart failure. These encouraging results provide a strong argument to move into first clinical trials. Already gene therapy for cardiac ischemia has reached human clinical trials, and early results of phase I clinical trials that employed adenoviral vectors to deliver either vascular endothelial growth factor (Ad-VEGF) or fibroblast growth factor 4 (Ad-FGF-4) to the ischemic heart showed no adverse events that could be attributed to adenovirus administration [12]. In this regard, bARKct gene therapy of human HF awaits application in the clinical arena. However, a major concern about bARK1 inhibition still stems from the notion that treatment of HF through enhancement, rather that supression of b-AR signaling, runs counter to current conventional wisdom in the clinical therapy of HF. This notion is based on the observation that application of b-AR agonists has been shown to be detrimental, whereas b-AR blockade has remarkable salutary effects in HF. It therefore seems important to note that b-blockade by carvedilol decreases the expression and activity of bARK1 in the hearts of mice www.sciencedirect.com
Vol.22 No.10 October 2004
chronically treated with this agent [13]. Thus, decreased bARK1 and resultant enhancement of cardiac b-AR signaling might represent, at least in part, a possible mechanism for the positive beneficial effect seen by b-AR blockade in HF. Moreover, Koch and colleagues have already demonstrated that cardiac bARK1 inhibition by bARKct, together with b-AR blockade by metoprolol, act synergistically to reduce lethal arrhythmias and promote favourable remodelling in HF [10]. This has been shown in a murine model of severe HF in which cardiac bARK1 inhibition prolongs survival and potentiated b-AR antagonist therapy [10] – an interesting finding given recent clinical results that show improved survival of patients with chronic HF that were treated with b-blockers. Therefore, these interventions appear to function synergistically rather than opposing each other and, so far, bARK1 inhibition cannot be explained by any existing pharmacological paradigm. However, it should be remembered that bARKct also functions more generally to sequester Gbg–subunits from other biologically important GPCRs (e.g. angiotensin II and endothelin receptors) that are involved in the activation of hypertrophic pathways, and there might be inhibition of other as yet unknown Gbg–effectors [3]. The extent (if any) to which interruption of such interactions might contribute to the salutary effects of bARKct remains to be determined. bARK1 inhibition in HF: a clinical perspective The current results provided by Koch and coworkers [1] clearly demonstrate a novel therapeutic principle in human failing myocardium that awaits extension to clinical practice. To achieve this objective, it will be necessary to make further advances in gene-delivery technology and vector design to effectively test this option in the clinical arena. Current gene-delivery methods remain quite invasive. Engineered vectors with tissue tropism (e.g. AAV) that obviate the need for direct myocardial delivery would represent a significant advance. Furthermore, research has not yielded any method to control expression after gene delivery. Analogous to a physician adjusting a dose to produce a desired effect, finer exogenous control of transgene expression would be desirable. Moreover, in addition to bARKct gene therapy, recent advances in the field of drug-development might facilitate the development of small-molecule inhibitors of bARK1 and its interaction with Gbg that might add new drugs to the therapeutic armamentarium for the treatment of heart failure. References 1 Williams, M.L. et al. (2004) Targeted b-adrenergic receptor kinase (bARK1) inhibition by gene transfer in failing human hearts. Circulation 109, 1590–1593 2 Rockman, H.A. et al. (2002) Seven-transmembrane-spanning receptors and heart function. Nature 415, 206–212 3 Koch, W.J. et al. (2000) Functional consequences of altering myocardial adrenergic receptor signaling. Annu. Rev. Physiol. 62, 237–260 4 Lefkowitz, R.J. et al. (2000) Catecholamines, cardiac beta-adrenergic receptors, and heart failure. Circulation 101, 1634–1637 5 Koch, W.J. et al. (1995) Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science 268, 1350–1353
Update
TRENDS in Biotechnology
6 Jaber, M. et al. (1996) Essential role of b-adrenergic receptor kinase-1 in cardiac development and function. Proc. Natl. Acad. Sci. U. S. A. 93, 12974–12979 7 Rockman, H.A. et al. (1998) Control of myocardial contractile function by the level of b-adrenergic receptor kinase-1 in gene-targeted mice. J. Biol. Chem. 273, 18180–18184 8 Ungerer, M. et al. (1993) Altered expression of b-adrenergic receptor kinase and b1-adrenergic receptors in the failing heart. Circulation 87, 454–463 9 Rockman, H.A. et al. (1998) Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc. Natl. Acad. Sci. U. S. A. 95, 7000–7005 10 Harding, V.B. et al. (2001) Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure. Proc. Natl. Acad. Sci. U. S. A. 98, 5809–5814
Vol.22 No.10 October 2004
489
11 Freeman, K. et al. (2001) Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J. Clin. Invest. 107, 967–974 12 Williams, M.L. and Koch, W.J. (2004) Viral-based myocardial gene therapy approaches to alter cardiac function. Annu. Rev. Physiol. 66, 49–75 13 Iaccarino, G. et al. (1998) Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by b-adrenergic receptor stimulation and blockade. Circulation 98, 1783–1789
0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.08.004
Tailor-made glycoproteins Linda C. Hsieh–Wilson Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
Posttranslational modifications are a fundamental mechanism for the regulation of cellular physiology and function. A recent paper by Zhang et al. provides a novel strategy for the generation of homogeneous glycoproteins. The ability to install covalent modifications site-specifically into proteins holds tremendous promise for deciphering the role of posttranslational modifications and has exciting implications for the development of protein therapeutics. Given that the complexity of higher organisms is encoded in a remarkably small number of genes, attention has focused on other mechanisms to account for the diversity of biological systems. Protein posttranslational modifications (PTMs) represent a major source of variation – the covalent attachment of phosphate, lipid, and other chemical groups to amino acid side chains extends the capabilities of proteins and provides selective and temporal control over protein function. At a cellular level, PTMs have been shown to regulate vital processes such as transcription, cell-cycle progression, and programmed cell death. Protein glycosylation, the most abundant and complex form of PTM, requires an estimated 1% of mammalian genes [1] and distinguishes eukaryotic cells from simpler prokaryotic systems. Recent studies are challenging early views of carbohydrates as non-specific, static structures and are revealing exciting physiological roles for glycosylation. For example, Wei et al. have shown that HIV evades the immune system by evolving a dynamically changing shield of carbohydrates [2]. Glycosaminoglycans, another class of carbohydrates, play crucial roles during development, with genetic disruption of Corresponding author: Linda C. Hsieh–Wilson ([email protected]). Available online 28 August 2004 www.sciencedirect.com
glycosaminoglycan biosynthesis associated with severe growth defects [3]. The complex sulfation patterns present in glycosaminoglycans, which are tissue-specific, tightly regulated, and crucial for growth factor activation, suggest an intricate relationship between carbohydrate structure and biological function [3]. In the intracellular context, dynamic forms of glycosylation have been identified, such as the addition of b-N-acetylglucosamine to serine or threonine residues of proteins (O-GlcNAc glycosylation) [4]. Recent studies have linked O-GlcNAc glycosylation to the regulation of transcription factors such as cAMP-responsive element binding protein (CREB) [5] and the proteasome [6], raising the exciting possibility that this carbohydrate modification plays a fundamental role in cellular communication and protein function. Despite many important contributions, the diverse roles of glycosylation and other covalent modifications are only beginning to be understood. Detailed studies of their biological effects have been hindered by the dynamic nature and complexity of PTMs in vivo. For instance, the presence of a given modification can depend on the cell type or status (e.g. stimulated vs non-stimulated, normal vs diseased). It can also be restricted to subcellular compartments or protein subpopulations and can occur in combination with other PTMs. Except for protein phosphorylation, in which an aspartic or glutamic acid can sometimes mimic the phosphorylated residue, no natural amino acid can serve as an effective surrogate for glycosylation and other PTMs. Moreover, efforts to purify modified proteins for biochemical studies generally require abundant sources and extensive purification. Even when available, the purified proteins are frequently heterogeneous, bearing incomplete or multiple modifications. A recent paper by Schultz and colleagues provides a novel solution to these problems and should considerably