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Intrinsic protein disorder and protein modifications in the processing of biological signals
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ScienceDirect Intrinsic protein disorder and protein modifications in the processing of biological signals Aaron H Phillips and Richard W Kriwacki Eukaryotic cells are highly complex systems; however, they manage to attain this complexity with a surprisingly small number of protein products. This is due, in part, to the fact that the functions of the eukaryotic proteome can be modulated and controlled by a vast network of largely reversible posttranslational modifications. Such modifications change the chemical nature of certain amino acid side chains and thereby can be used to modulate diverse protein functions such as enzyme activity and binding events. Here we review recent advances in the characterization of the native mechanisms by which cells utilize post-translational modifications to send biological signals as well as recent successes in engineering such systems. We highlight roles for protein disorder in signal propagation in these systems. Address Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, United States Corresponding author: Kriwacki, Richard W ([email protected])
Current Opinion in Structural Biology 2020, 60:1–6 This review comes from a themed issue on Folding and binding Edited by Shachi Gosavi and Ben Schuler
https://doi.org/10.1016/j.sbi.2019.09.003 0959-440X/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Post-translational modifications (PTMs) of proteins are often used as conduits for the flow of biological information and play key roles in signaling cascades that control critical cellular decisions such as whether to divide or execute programmed cell death. Misregulation of these processes underlie many human diseases and over the past few decades great advances have been made in mapping and even pharmacologically perturbing these signaling pathways. In recent years these advances in understanding the basic mechanisms by which cells process information have led to increasing interest in utilizing this machinery to create novel, engineered signaling circuits in the burgeoning field of synthetic biology. In this review we highlight several recent studies that exemplify how post-translational modifications, frequently www.sciencedirect.com
within disordered regions of proteins, are utilized in cell signaling, both within native biological systems as well as in novel synthetic circuits. The mediation of biological signaling by the post-translational modification of proteins is an admittedly vast topic and many aspects of the field are outside the scope of this review. For discussion of the following topics we direct the reader to excellent reviews focused on each. Protein localization can be restricted to the membrane via protein lipidation [1] and the function of proteins is often modulated via the addition of sugar moieties [2]. The disordered tails of histones are known to be heavily post-translationally modified, and the resulting epigenetic status of histones controls the expression of nearby genes [3–5]. Protein homeostasis is controlled via modification with ubiquitin [6] and protein modification with ubiquitin chains can also mediate myriad non-proteolytic signaling events [7]. Protein function can also be modulated by the addition of SUMO [8] or NEDD8 [9]. Lastly, proteolysis cascades are crucial for the regulation of processes such as apoptosis and coagulation [10–12]. Signaling proteins are enriched in intrinsically disordered regions (IDRs) and many such Sic1 and p27, are completely disordered in solution [13]. Disorder in these proteins provides numerous advantages in the processing of biological signals. Disordered regions contain a higher density of amino acids that can be targeted by PTMs [14], present their entire sequence that can contain many short linear motifs (SLiMs) for potential binding partners and enzymes [15], and are easily targeted by alternative splicing events without disruption of global protein folds [16,17]. Moreover, crosstalk between alternative splicing and PTMs within intrinsically disordered regions can be harnessed for increased signaling complexity [18] and conceptually mirrors the increased signaling complexity that can be achieved through crosstalk between multiple PTMs as observed with the numerous epigenetic marks that occur within histones [19–21].
Lysine modification outside histones The amino side chain of lysine is a frequent PTM target and is commonly observed to be acetylated or methylated with emerging evidence that these PTMs can mediate biological functions outside epigenetics. For example, the activity of metabolic enzymes is regulated by lysine acetylation in a process that is conserved from prokaryotes to eukaryotes [22,23]. Central metabolic enzymes in Salmonella were shown to be differentially acetylated in response to growth on different carbon sources, controlling the relative flux Current Opinion in Structural Biology 2020, 60:1–6
2 Folding and binding
Arginine side chains can also undergo methylation, which alters the non-covalent interaction potential of the modified guanidinium moiety. Arginine methylation of PTEN modulates the activity of the PI3K–Akt pathway, which is often misregulated in cancers [29]. Recent studies have found that this type of modification plays roles in the formation of biomolecular condensates such as those formed by FUS that play roles in amyotrophic lateral sclerosis and other neurodegenerative disorders [30,31]. The N-terminal domain of AGO2, a core component of the RNAi machinery, is enriched in RG/ GR repeats that have been shown to be methylated by PRMT5, promoting the degradation of AGO2 and AGO2associated small RNAs [32]. Given the prominent role of RG/GR repeats in proteins that are prone to phase separation [33], it is tempting to speculate that effects of methylation on AGO2 regulation could be linked to phase separation phenomena.
the formation of pathogenic cis phospho-tau aggregates in models of Alzheimer’s disease [39]. Pin1 has also been shown to enhance p53-dependent BAX activation by catalyzing the isomerization of proline 47 within p53, triggering pro-apoptotic conformational changes in BAX [40]. The transactivation domain of the circadian clock regulator BMAL1 contains a highly conserved tryptophan-proline pair, the isomerization of which controls the timing of circadian periods [41]. The isomerization at a conserved proline residue in the nuclear-coactivator binding domain of CBP modulates the kinetics of association with its binding partner ACTR [42]. Such a switching behavior encoded in the IDRs of protein machines allows for dynamic and reversible control critical biological processes, as has been observed for control of transcription by RNA Polymerase II (RNA Pol II). The functional state of this essential enzyme is regulated by PTMs within its C-terminal IDR which, in humans, comprised 52 repeats of a heptad amino acid sequence, including Serine 5 and Proline 6. For example, the isomerization status of Proline 6, which is in turn regulated by phosphorylation status of Serine 5, directs the activity of the phosphatase Ssu72 that is important for transcriptional termination [43]. Recently, it was shown that, before termination, phosphorylation of residues within the IDR by CDK7 and CDK9 mediates differential phase separation of RNA Pol II with components of the transcriptional machinery that mediate the transition from initiation to elongation [44]. However, the role of interplay between phosphorylation of Serine and Threonine residues within the heptad repeats and Proline isomerization was not probed in the latter study.
Asparagine deamidation
Phosphorylation mediated signal integration
New research is illuminating how viral machinery can deamidate host proteins to interfere with the host immune response [34]. The Salmonella effector protein, SseI, deamidates host heterotrimeric Gi proteins, resulting in their persistent activation and subsequent upregulation of pro-survival pathways [35]. Recent studies have shown that the resistance of some non-human primates to HSV-1 infection is due to the variation of a single asparagine residue within the innate immune sensor cGAS, which is deamidated by the UL37 viral effector [36]. Deamidation of native eukaryotic proteins has also been shown to modulate apoptotic signaling. Bcl-xL contains a long IDR that is a hot spot for PTMs, including sites for both phosphorylation and deamidation, that can tune apoptotic signaling [37].
Phosphorylation is the most commonly observed PTM of proteins [45]. Phosphorylation cascades are essential signaling events and have been intensely studied. Proteins involved in signaling often contain IDRs that are enriched in SLiMs that are recognized by kinases, phosphatases, and phospho-binding domains that function, respectively, as the writers, erasers, and readers of phosphorylation-based biological information. Two archetypal disordered signal integrating proteins are the cyclindependent kinase (Cdk) inhibitors, Sic1 (from yeast) and p27 (from humans) (Figure 1). These proteins, which, before their phosphorylation, bind and potently inhibit Cdks, serve to integrate inputs from pro-mitotic stimuli and then allow for progression through the cell cycle after being selectively degraded, thereby releasing the inhibitory brake on Cdk activity. Upon pro-mitotic activation of nonreceptor tyrosine kinases (NRTKs) such as Abl or Src, p27 is phosphorylated at Y88 and/or Y74, ejecting these inhibitory residues from the active site of Cdk2, thereby restoring partial activity to the kinase. Partially active Cdk2 in turn phosphorylates Threonine 187 on p27 through a pseudo-unimolecular intra-complex mechanism, thus forming the phosphodegron that
through the citric acid and glyoxylate cycles [22]. Similar dynamic control of metabolic flux was demonstrated in the human liver where metabolic enzymes are differentially acetylated in response to the concentration of metabolic fuels such as glucose, amino acids, and fatty acids [23]. Autophagy is also known to be regulated by the lysine acetylation status of enzymes such as Atg3 and Ulk1 [24,25]. MAP3K2 has been shown to be methylated in a Ras-driven cancer [26] and the activity of Akt is stimulated by lysine methylation mediated by SETDB1, a new potential target for the development of anti-cancer therapeutics [27,28].
Arginine methylation
Proline isomerization Disordered regions of proteins are often enriched in proline residues [38], which can populate the cis or trans isomers about the prolyl peptide bond. Interconversion between these two isomers can occur spontaneously and is also catalyzed by the peptidyl-prolyl isomerases (PPIases). Pin1 is a PPIase that has been shown to prevent Current Opinion in Structural Biology 2020, 60:1–6
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Disorder and PTMs in biological signal processing Phillips and Kriwacki 3
Figure 1
Cdk2 is inhibited
SCFSkp2 recruitment
c Sr Cdk2 ~50% active
Ab
p27 degradation
I
Cdk2 100% active Cdk2 ~20% active Current Opinion in Structural Biology
Progression through the mammalian cell division cycle is triggered by the activation of non-receptor tyrosine kinases such as Src and Abl which phosphorylate p27, causing ejection of the tyrosine residues from the Cdk2 active site, ultimately leading to selective degradation of the inhibitor. The Cdk2/cyclin A/p27 complex integrates the differential phosphorylation patterns depending on the substrate specificity of the active tyrosine kinase. Activation of kinases that phosphorylate both tyrosine 74 and 88, such as Src, result in a stronger pro-mitotic signal.
recruits the SCFSkp2 E3 ubiquitin ligase that targets p27 for degradation. Elimination of p27 yields a full active Cdk2/cyclin A complex that can then phosphorylate downstream substrates, driving cell cycle progression [46,47]. Yeast do not encode tyrosine kinases and therefore cannot regulate the cell cycle using orthogonal Tyrosine and Serine/Threonine kinases and have evolved a processive, threshold mechanism where the phosphorylation of CDK complex inhibitors such as Sic1 is directed from its N-terminus to C-terminus by molecular handoffs between Cks1, Cdk1, and cyclins encoded by motifs present in their amino acid sequence [48]. Only when inhibitors such as Sic1 have been sufficiently phosphorylated are they recognized by the SCFCdc4 E3 ligase complex, targeting them for proteasomal degradation and thereby progressing to the next step of the cell cycle [49,50]. In yeast a similar threshold mechanism is used to target Eco1 for degradation only after it has been
phosphorylated by three kinases with distinct specificities, functioning as an AND logic gate [51]. Additional, recent examples of such phosphorylation-based signal processors include the binary switch PAGE4 which switches from an activator of cJun to an attenuator based on the substrate specificity of the kinase that phosphorylates PAGE4 [52] and the phosphorylation of PKS4 was recently shown to switch off phototropism in plants [53]. Phosphorylation switches have also been found to control disassembly of membraneless organelles such as has been observed with Casein kinase 2 promoting the dissolution of stress granules, likely via phosphorylation of the stress granule nucleating protein G3BP1 [54]. These types of sensors and switches can conceivably be engineered to create novel biological circuits. Since the logic occurs at the protein level rather than at the level of gene activation, this approach can ideally result in much
Figure 2
WD40
Fus3
protein X induce proximity dependent property with α-factor
protein Y docking peptide
substrate Current Opinion in Structural Biology
Schematic depiction of the engineered phospho-regulon system described by Gordley et al. Upon stimulation with a-factor, Fus3 phosphorylates the synthetic phospho-regulon which then recruits a WD40 domain, thereby bringing protein X and protein Y into close proximity. www.sciencedirect.com
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4 Folding and binding
demonstrated the use of this engineered phosphorregulon to induce membrane localization, target a substrate for degradation, and generate a positive feedback loop upon Fus3 activation [55] (Figure 2).
Figure 3
(a)
inactive protease active protease
blocked scaffold
(b)
TEVP
Synthetic circuits based on protease engineering activated scaffold degron TEVP substrate HCVP substrate OR
luciferase on
AND CFP
HCVP
(c)
split protease
inducer
subatrate inhibitory coil
split reporter
reporter on Current Opinion in Structural Biology
Synthetic biological circuits based on protease engineering. (a) Split luciferase is reconstituted on a caspase-9 activatable scaffold based on 14-3-3. (b) Example of logic gates consisting of proteases from the CHOMP (circuits of hacked orthogonal modular proteases) system. Tobacco etch virus protease (TEVP) and hepatitis C virus protease (HCVP) were used as the basis for the construction of citrine fluorescent protein (CFP)-based reporter systems. In the absence of active proteases, the degron sequence causes destruction of the CFP constructs. In the ‘OR’ logic gate activation of either protease removes the degron, thereby stabilizing CFP. In the ‘AND’ gate example, the activity of both proteases is required to stabilize CFP. Cartoon helices indicate complementary leucine zippers that were used to increase the protease specificity. (c) Schematic depiction of the split protease cleavable orthogonal coiled coil-based (SPOC) logic circuits. Split proteases are activated by assembly with an inducer. The activated protease then cleaves off the inhibitory coiled coil that blocks assembly of the split luciferase reporter.
faster logic operations in synthetic applications. Early efforts to engineer these types of circuits utilized the splicing of engineered recruitment interactions and SLiM motifs into pre-existing networks [55,56]. Specifically, Gordley et al. engineered a synthetic phospho-regulon to recruit the yeast mitogen-activated protein kinase, Fus3 to phosphorylate the Tec1 substrate site, which in turn triggers binding of a WD40 domain. The authors Current Opinion in Structural Biology 2020, 60:1–6
Another approach to engineer protein-level logic circuits is based on protease-based signaling networks inspired by caspase activation and coagulation cascades. Several labs have recently reported success with this strategy (Figure 3). The Merkx lab reported a system in which cleavable inhibitory peptides, fused to 14-3-3, could be removed upon activation of a dimerization controllable caspase 9. Upon removal of the inhibitory peptides, 14-3-3 could then template the formation of an active luciferase from split luciferase subunits [57]. Groundbreaking work from Michaek Ekowitz’s lab has demonstrated that viral proteases combined with synthetic degrons and oligomerization sequences can function as composable protein components of synthetic circuits. They termed this system CHOMP for circuits of hacked orthogonal modular proteases and showed that these components can be assembled to form logic gates and create regulatory cascades [58]. Soon after, the Jerala group reported similar results based on split proteases that can be reassembled using engineered coiled coil domains and termed this design as SPOC logic circuits for split-protease-cleavable orthogonal-coiled coils [59]. In the reports describing the CHOMP and SPOC logic systems, the authors demonstrate that these synthetic circuits can perform a complete set of Boolean logic operations, representing a groundbreaking achievement in the development of synthetic biological systems.
Concluding remarks The post-translational modification of proteins has been harnessed by evolution to produce a wide variety of natural signaling events, playing critical roles in all kingdoms of life. One of the most striking insights from the genomic era has been that the increased complexity in higher organisms is achieved with a surprisingly small number of protein products. Higher organisms have attained their complexity, in part, by utilizing the signaling versatility afforded by intrinsic disorder and post-translational modifications to form multistep and multifunctional signaling networks. Inspired by these natural signaling systems, synthetic biologists will surely continue to innovate new protein-based signaling circuits for the development of novel biotechnologies.
Conflict of interest statement Nothing declared.
Acknowledgement The authors thank ALSAC for funding. www.sciencedirect.com
Disorder and PTMs in biological signal processing Phillips and Kriwacki 5
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ScienceDirect Intrinsic protein disorder and protein modifications in the processing of biological signals Aaron H Phillips and Richard W Kriwacki Eukaryotic cells are highly complex systems; however, they manage to attain this complexity with a surprisingly small number of protein products. This is due, in part, to the fact that the functions of the eukaryotic proteome can be modulated and controlled by a vast network of largely reversible posttranslational modifications. Such modifications change the chemical nature of certain amino acid side chains and thereby can be used to modulate diverse protein functions such as enzyme activity and binding events. Here we review recent advances in the characterization of the native mechanisms by which cells utilize post-translational modifications to send biological signals as well as recent successes in engineering such systems. We highlight roles for protein disorder in signal propagation in these systems. Address Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, United States Corresponding author: Kriwacki, Richard W ([email protected])
Current Opinion in Structural Biology 2020, 60:1–6 This review comes from a themed issue on Folding and binding Edited by Shachi Gosavi and Ben Schuler
https://doi.org/10.1016/j.sbi.2019.09.003 0959-440X/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Post-translational modifications (PTMs) of proteins are often used as conduits for the flow of biological information and play key roles in signaling cascades that control critical cellular decisions such as whether to divide or execute programmed cell death. Misregulation of these processes underlie many human diseases and over the past few decades great advances have been made in mapping and even pharmacologically perturbing these signaling pathways. In recent years these advances in understanding the basic mechanisms by which cells process information have led to increasing interest in utilizing this machinery to create novel, engineered signaling circuits in the burgeoning field of synthetic biology. In this review we highlight several recent studies that exemplify how post-translational modifications, frequently www.sciencedirect.com
within disordered regions of proteins, are utilized in cell signaling, both within native biological systems as well as in novel synthetic circuits. The mediation of biological signaling by the post-translational modification of proteins is an admittedly vast topic and many aspects of the field are outside the scope of this review. For discussion of the following topics we direct the reader to excellent reviews focused on each. Protein localization can be restricted to the membrane via protein lipidation [1] and the function of proteins is often modulated via the addition of sugar moieties [2]. The disordered tails of histones are known to be heavily post-translationally modified, and the resulting epigenetic status of histones controls the expression of nearby genes [3–5]. Protein homeostasis is controlled via modification with ubiquitin [6] and protein modification with ubiquitin chains can also mediate myriad non-proteolytic signaling events [7]. Protein function can also be modulated by the addition of SUMO [8] or NEDD8 [9]. Lastly, proteolysis cascades are crucial for the regulation of processes such as apoptosis and coagulation [10–12]. Signaling proteins are enriched in intrinsically disordered regions (IDRs) and many such Sic1 and p27, are completely disordered in solution [13]. Disorder in these proteins provides numerous advantages in the processing of biological signals. Disordered regions contain a higher density of amino acids that can be targeted by PTMs [14], present their entire sequence that can contain many short linear motifs (SLiMs) for potential binding partners and enzymes [15], and are easily targeted by alternative splicing events without disruption of global protein folds [16,17]. Moreover, crosstalk between alternative splicing and PTMs within intrinsically disordered regions can be harnessed for increased signaling complexity [18] and conceptually mirrors the increased signaling complexity that can be achieved through crosstalk between multiple PTMs as observed with the numerous epigenetic marks that occur within histones [19–21].
Lysine modification outside histones The amino side chain of lysine is a frequent PTM target and is commonly observed to be acetylated or methylated with emerging evidence that these PTMs can mediate biological functions outside epigenetics. For example, the activity of metabolic enzymes is regulated by lysine acetylation in a process that is conserved from prokaryotes to eukaryotes [22,23]. Central metabolic enzymes in Salmonella were shown to be differentially acetylated in response to growth on different carbon sources, controlling the relative flux Current Opinion in Structural Biology 2020, 60:1–6
2 Folding and binding
Arginine side chains can also undergo methylation, which alters the non-covalent interaction potential of the modified guanidinium moiety. Arginine methylation of PTEN modulates the activity of the PI3K–Akt pathway, which is often misregulated in cancers [29]. Recent studies have found that this type of modification plays roles in the formation of biomolecular condensates such as those formed by FUS that play roles in amyotrophic lateral sclerosis and other neurodegenerative disorders [30,31]. The N-terminal domain of AGO2, a core component of the RNAi machinery, is enriched in RG/ GR repeats that have been shown to be methylated by PRMT5, promoting the degradation of AGO2 and AGO2associated small RNAs [32]. Given the prominent role of RG/GR repeats in proteins that are prone to phase separation [33], it is tempting to speculate that effects of methylation on AGO2 regulation could be linked to phase separation phenomena.
the formation of pathogenic cis phospho-tau aggregates in models of Alzheimer’s disease [39]. Pin1 has also been shown to enhance p53-dependent BAX activation by catalyzing the isomerization of proline 47 within p53, triggering pro-apoptotic conformational changes in BAX [40]. The transactivation domain of the circadian clock regulator BMAL1 contains a highly conserved tryptophan-proline pair, the isomerization of which controls the timing of circadian periods [41]. The isomerization at a conserved proline residue in the nuclear-coactivator binding domain of CBP modulates the kinetics of association with its binding partner ACTR [42]. Such a switching behavior encoded in the IDRs of protein machines allows for dynamic and reversible control critical biological processes, as has been observed for control of transcription by RNA Polymerase II (RNA Pol II). The functional state of this essential enzyme is regulated by PTMs within its C-terminal IDR which, in humans, comprised 52 repeats of a heptad amino acid sequence, including Serine 5 and Proline 6. For example, the isomerization status of Proline 6, which is in turn regulated by phosphorylation status of Serine 5, directs the activity of the phosphatase Ssu72 that is important for transcriptional termination [43]. Recently, it was shown that, before termination, phosphorylation of residues within the IDR by CDK7 and CDK9 mediates differential phase separation of RNA Pol II with components of the transcriptional machinery that mediate the transition from initiation to elongation [44]. However, the role of interplay between phosphorylation of Serine and Threonine residues within the heptad repeats and Proline isomerization was not probed in the latter study.
Asparagine deamidation
Phosphorylation mediated signal integration
New research is illuminating how viral machinery can deamidate host proteins to interfere with the host immune response [34]. The Salmonella effector protein, SseI, deamidates host heterotrimeric Gi proteins, resulting in their persistent activation and subsequent upregulation of pro-survival pathways [35]. Recent studies have shown that the resistance of some non-human primates to HSV-1 infection is due to the variation of a single asparagine residue within the innate immune sensor cGAS, which is deamidated by the UL37 viral effector [36]. Deamidation of native eukaryotic proteins has also been shown to modulate apoptotic signaling. Bcl-xL contains a long IDR that is a hot spot for PTMs, including sites for both phosphorylation and deamidation, that can tune apoptotic signaling [37].
Phosphorylation is the most commonly observed PTM of proteins [45]. Phosphorylation cascades are essential signaling events and have been intensely studied. Proteins involved in signaling often contain IDRs that are enriched in SLiMs that are recognized by kinases, phosphatases, and phospho-binding domains that function, respectively, as the writers, erasers, and readers of phosphorylation-based biological information. Two archetypal disordered signal integrating proteins are the cyclindependent kinase (Cdk) inhibitors, Sic1 (from yeast) and p27 (from humans) (Figure 1). These proteins, which, before their phosphorylation, bind and potently inhibit Cdks, serve to integrate inputs from pro-mitotic stimuli and then allow for progression through the cell cycle after being selectively degraded, thereby releasing the inhibitory brake on Cdk activity. Upon pro-mitotic activation of nonreceptor tyrosine kinases (NRTKs) such as Abl or Src, p27 is phosphorylated at Y88 and/or Y74, ejecting these inhibitory residues from the active site of Cdk2, thereby restoring partial activity to the kinase. Partially active Cdk2 in turn phosphorylates Threonine 187 on p27 through a pseudo-unimolecular intra-complex mechanism, thus forming the phosphodegron that
through the citric acid and glyoxylate cycles [22]. Similar dynamic control of metabolic flux was demonstrated in the human liver where metabolic enzymes are differentially acetylated in response to the concentration of metabolic fuels such as glucose, amino acids, and fatty acids [23]. Autophagy is also known to be regulated by the lysine acetylation status of enzymes such as Atg3 and Ulk1 [24,25]. MAP3K2 has been shown to be methylated in a Ras-driven cancer [26] and the activity of Akt is stimulated by lysine methylation mediated by SETDB1, a new potential target for the development of anti-cancer therapeutics [27,28].
Arginine methylation
Proline isomerization Disordered regions of proteins are often enriched in proline residues [38], which can populate the cis or trans isomers about the prolyl peptide bond. Interconversion between these two isomers can occur spontaneously and is also catalyzed by the peptidyl-prolyl isomerases (PPIases). Pin1 is a PPIase that has been shown to prevent Current Opinion in Structural Biology 2020, 60:1–6
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Disorder and PTMs in biological signal processing Phillips and Kriwacki 3
Figure 1
Cdk2 is inhibited
SCFSkp2 recruitment
c Sr Cdk2 ~50% active
Ab
p27 degradation
I
Cdk2 100% active Cdk2 ~20% active Current Opinion in Structural Biology
Progression through the mammalian cell division cycle is triggered by the activation of non-receptor tyrosine kinases such as Src and Abl which phosphorylate p27, causing ejection of the tyrosine residues from the Cdk2 active site, ultimately leading to selective degradation of the inhibitor. The Cdk2/cyclin A/p27 complex integrates the differential phosphorylation patterns depending on the substrate specificity of the active tyrosine kinase. Activation of kinases that phosphorylate both tyrosine 74 and 88, such as Src, result in a stronger pro-mitotic signal.
recruits the SCFSkp2 E3 ubiquitin ligase that targets p27 for degradation. Elimination of p27 yields a full active Cdk2/cyclin A complex that can then phosphorylate downstream substrates, driving cell cycle progression [46,47]. Yeast do not encode tyrosine kinases and therefore cannot regulate the cell cycle using orthogonal Tyrosine and Serine/Threonine kinases and have evolved a processive, threshold mechanism where the phosphorylation of CDK complex inhibitors such as Sic1 is directed from its N-terminus to C-terminus by molecular handoffs between Cks1, Cdk1, and cyclins encoded by motifs present in their amino acid sequence [48]. Only when inhibitors such as Sic1 have been sufficiently phosphorylated are they recognized by the SCFCdc4 E3 ligase complex, targeting them for proteasomal degradation and thereby progressing to the next step of the cell cycle [49,50]. In yeast a similar threshold mechanism is used to target Eco1 for degradation only after it has been
phosphorylated by three kinases with distinct specificities, functioning as an AND logic gate [51]. Additional, recent examples of such phosphorylation-based signal processors include the binary switch PAGE4 which switches from an activator of cJun to an attenuator based on the substrate specificity of the kinase that phosphorylates PAGE4 [52] and the phosphorylation of PKS4 was recently shown to switch off phototropism in plants [53]. Phosphorylation switches have also been found to control disassembly of membraneless organelles such as has been observed with Casein kinase 2 promoting the dissolution of stress granules, likely via phosphorylation of the stress granule nucleating protein G3BP1 [54]. These types of sensors and switches can conceivably be engineered to create novel biological circuits. Since the logic occurs at the protein level rather than at the level of gene activation, this approach can ideally result in much
Figure 2
WD40
Fus3
protein X induce proximity dependent property with α-factor
protein Y docking peptide
substrate Current Opinion in Structural Biology
Schematic depiction of the engineered phospho-regulon system described by Gordley et al. Upon stimulation with a-factor, Fus3 phosphorylates the synthetic phospho-regulon which then recruits a WD40 domain, thereby bringing protein X and protein Y into close proximity. www.sciencedirect.com
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4 Folding and binding
demonstrated the use of this engineered phosphorregulon to induce membrane localization, target a substrate for degradation, and generate a positive feedback loop upon Fus3 activation [55] (Figure 2).
Figure 3
(a)
inactive protease active protease
blocked scaffold
(b)
TEVP
Synthetic circuits based on protease engineering activated scaffold degron TEVP substrate HCVP substrate OR
luciferase on
AND CFP
HCVP
(c)
split protease
inducer
subatrate inhibitory coil
split reporter
reporter on Current Opinion in Structural Biology
Synthetic biological circuits based on protease engineering. (a) Split luciferase is reconstituted on a caspase-9 activatable scaffold based on 14-3-3. (b) Example of logic gates consisting of proteases from the CHOMP (circuits of hacked orthogonal modular proteases) system. Tobacco etch virus protease (TEVP) and hepatitis C virus protease (HCVP) were used as the basis for the construction of citrine fluorescent protein (CFP)-based reporter systems. In the absence of active proteases, the degron sequence causes destruction of the CFP constructs. In the ‘OR’ logic gate activation of either protease removes the degron, thereby stabilizing CFP. In the ‘AND’ gate example, the activity of both proteases is required to stabilize CFP. Cartoon helices indicate complementary leucine zippers that were used to increase the protease specificity. (c) Schematic depiction of the split protease cleavable orthogonal coiled coil-based (SPOC) logic circuits. Split proteases are activated by assembly with an inducer. The activated protease then cleaves off the inhibitory coiled coil that blocks assembly of the split luciferase reporter.
faster logic operations in synthetic applications. Early efforts to engineer these types of circuits utilized the splicing of engineered recruitment interactions and SLiM motifs into pre-existing networks [55,56]. Specifically, Gordley et al. engineered a synthetic phospho-regulon to recruit the yeast mitogen-activated protein kinase, Fus3 to phosphorylate the Tec1 substrate site, which in turn triggers binding of a WD40 domain. The authors Current Opinion in Structural Biology 2020, 60:1–6
Another approach to engineer protein-level logic circuits is based on protease-based signaling networks inspired by caspase activation and coagulation cascades. Several labs have recently reported success with this strategy (Figure 3). The Merkx lab reported a system in which cleavable inhibitory peptides, fused to 14-3-3, could be removed upon activation of a dimerization controllable caspase 9. Upon removal of the inhibitory peptides, 14-3-3 could then template the formation of an active luciferase from split luciferase subunits [57]. Groundbreaking work from Michaek Ekowitz’s lab has demonstrated that viral proteases combined with synthetic degrons and oligomerization sequences can function as composable protein components of synthetic circuits. They termed this system CHOMP for circuits of hacked orthogonal modular proteases and showed that these components can be assembled to form logic gates and create regulatory cascades [58]. Soon after, the Jerala group reported similar results based on split proteases that can be reassembled using engineered coiled coil domains and termed this design as SPOC logic circuits for split-protease-cleavable orthogonal-coiled coils [59]. In the reports describing the CHOMP and SPOC logic systems, the authors demonstrate that these synthetic circuits can perform a complete set of Boolean logic operations, representing a groundbreaking achievement in the development of synthetic biological systems.
Concluding remarks The post-translational modification of proteins has been harnessed by evolution to produce a wide variety of natural signaling events, playing critical roles in all kingdoms of life. One of the most striking insights from the genomic era has been that the increased complexity in higher organisms is achieved with a surprisingly small number of protein products. Higher organisms have attained their complexity, in part, by utilizing the signaling versatility afforded by intrinsic disorder and post-translational modifications to form multistep and multifunctional signaling networks. Inspired by these natural signaling systems, synthetic biologists will surely continue to innovate new protein-based signaling circuits for the development of novel biotechnologies.
Conflict of interest statement Nothing declared.
Acknowledgement The authors thank ALSAC for funding. www.sciencedirect.com
Disorder and PTMs in biological signal processing Phillips and Kriwacki 5
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