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Protein-protein interactions, or PPIs, are a part of every biological activity and have been linked to a number of diseases, including cancer, infectious diseases, and neurological disorders. As such, targeting PPIs is considered a strategic and vital approach in the development of new medications. Nonetheless, the wide and flat contact interface makes it difficult to find small-molecule PP inhibitors. An alternative strategy would be to use the PPI interaction motifs as building blocks for the design of peptide-based inhibitors. Herein, we designed 12-mer peptide inhibitors to target p25-inducing-cyclin-dependent kinase (Cdk5) hyperregulation, a PPI that has been shown to perpetuate neuroinflammation, which is one of the major causal implications of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and frontotemporal dementia. We generated a library of 5 062 500 peptide combination sequences (PCS) derived from the interaction motif of Cdk5/p25 PP interface. The 20 amino acids were differentiated into six groups, namely, hydrophobic (aliphatic), aromatic, basic, acidic, unique, and polar uncharged, on the basis of their physiochemical properties. To preserve the interaction motif necessary for ideal binding, de novo modeling of all possible peptide sequence substitutions was considered. A set of filters, backed by the Support Vector Machine (SVM) algorithm, was then used to create a shortlisted custom peptide library that met specific bioavailability, toxicity, and therapeutic relevance, leading to a refined library of 15 PCS. A greedy algorithm and coarse-grained force field were used to predict peptide structure and folding before subsequent modeling studies. Molecular docking was performed to estimate the relative binding affinities, and out of the top hits, Pep15 was subjected to molecular dynamics simulations and binding free-energy calculations in comparison to a known peptide inhibitor with experimental data (template peptide). Interestingly, the identified peptide through our protocol, Pep15, was found to show a significantly higher binding affinity than the reference template peptide (-48.10 ± 0.23 kcal/mol and -17.53 ± 0.27 kcal/mol, respectively). In comparison to the template peptide, Pep15 was found to possess a more compact and buried surface area, tighter binding landscape, and reduced conformational variability, leading to enhanced structural and kinetic stability of the Cdk5/p25 complex. Notably, both peptide inhibitors were found to have a minimal impact on the architectural integrity of the Cdk5/p25 secondary structure. Herein, we propose Pep15 as a novel and potentially disruptive peptide drug for Cdk5/p25-mediated neurodegenerative phenotypes that require further clinical investigation. The systematic protocol and findings of this report would serve as a valuable tool in the identification of critical PPI interface reactive residues, designing of analogs, and identification of more potent peptide-based PPI inhibitors., (© 2024 The Author(s). Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

Background: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology., Methods: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex., Results: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model., Conclusions: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease., (© 2024. The Author(s).)

The cyclin-dependent kinase (CDK5) forms a stable complex with its activator p25, leading to the hyperphosphorylation of tau proteins and to the formation of plaques and tangles that are considered to be one of the typical causes of Alzheimer's disease (AD). Hence, the pathological CDK5-p25 complex is a promising therapeutic target for AD. Small peptides, obtained from the truncation of CDK5 physiological activator p35, have shown promise in inhibiting the pathological complex effectively while also crossing the blood-brain barrier. One such small 24-residue peptide, p5, has shown selective inhibition toward the pathological complex in vivo. Our previous research focused on the characterization of a computationally predicted CDK5-p5 binding mode and of its pharmacophore, which was consistent with competitive inhibition. In continuation of our previous work, herein, we investigate four additional binding modes to explore other possible mechanisms of interaction between CDK5 and p5. The quantitative description of the pharmacophore is consistent with both competitive and allosteric p5-induced inhibition mechanisms of CDK5-p25 pathology. The gained insights can direct further in vivo/in vitro tests and help design small peptides, linear or cyclic, or peptidomimetic compounds as adjuvants of orthosteric inhibitors or as part of a cocktail of drugs with enhanced effectiveness and lower side effects.

Meiosis is a specialized cell division process that mediates genetic information transfer to the next generation. Meiotic chromosomal segregation occurs when DNA replication is completed during the pre-meiotic S phase. Here, we show that Schizosaccharomyces pombe Pef1, an orthologue of mammalian cyclin-dependent kinase 5 (CDK5), is required to promote pre-meiotic DNA replication. We examined the efficiency of meiotic initiation using pat1-114 mutants and found that, meiotic nuclear divisions did not occur in the pef1Δ pat1-114 strain. Deletion of pef1 also suppressed the expression of DNA replication factors and the phosphorylation of Cdc2 Tyr-15. The double deletion of clg1 and psl1 arrested meiotic initiation in pat1-114 mutant cells, similar to that of pef1 -deficient cells. Meiotic progression was also slightly delayed in the pas1 -deficient strain. Our results reveal that Pef1 regulates cyclin-coordinated meiotic progression.

Adiponectin is an adipocyte-derived cytokine having an insulin-sensitizing activity. During the phenotypic screening of secondary metabolites derived from the marine fungus Aspergillus terreus , a poly cyclin-dependent kinase (CDK) inhibitor butyrolactone I affecting CDK1 and CDK5 was discovered as a potent adiponectin production-enhancing compound in the adipogenesis model of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). CDK5 inhibitors exhibit insulin-sensitizing activities by suppressing the phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ). However, the adiponectin production-enhancing activities of butyrolactone I have not been correlated with the potency of CDK5 inhibitor activities. In a target identification study, butyrolactone I was found to directly bind to PPARγ. In the crystal structure of the human PPARγ, the ligand-binding domain (LBD) in complex with butyrolactone I interacted with the amino acid residues located in the hydrophobic binding pockets of the PPARγ LBD, which is a typical binding mode of the PPARγ partial agonists. Therefore, the adiponectin production-enhancing effect of butyrolactone I was mediated by its polypharmacological dual modulator activities as both a CDK5 inhibitor and a PPARγ partial agonist., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and characterized by brain cell death, memory loss and is the most common form of dementia. Although AD has devastating effects, however, drugs which can treat the AD remain limited. The cyclin-dependent kinase 5 (CDK5) has been recognized as being involved in the pathological hyperphosphorylation of tau protein, which leads to the formation of neurofibrillary tangles (NFTs). We utilized the structure-based virtual screening (SBVS) approach to find the potential inhibitors against HsCDK5. The natural compound subset from the ZINC database ( n  = 167,741) was retrieved and screened by using SBVS method. From here, we have predicted 297 potent inhibitors. These 297 compounds were evaluated through their pharmacokinetic properties by ADMET (absorption, distribution, metabolism, elimination/excretion and toxicity) descriptors. Finally, 17 compounds were selected and used for re-docking. After the refinement by molecular docking and by using drug-likeness analysis, we have identified four potential inhibitors (ZINC85877721, ZINC96114862, ZINC96115616 and ZINC96116231). All these four ligands were employed for 100 ns MDS study. From the root mean square deviation (RMSD), root mean square fluctuation (RMSF), Rg, number of hydrogen bonds, solvent accessible surface area (SASA), principal component analysis (PCA) and binding free energy analysis we have found that out of four inhibitors ZINC85877721 and ZINC96116231 showed good binding free energy of -198.84 and -159.32 kJ.mol -1 , respectively, and also good in other structural analyses. Both compounds displayed excellent pharmacological and structural properties to be the drug candidates. Collectively, these findings recommend that two compounds have great potential to be a promising agent against AD to reduce the CDK5 induced hyperphosphorylation and could be considered as therapeutic agents for the AD.Communicated by Ramaswamy H. Sarma.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A new series of derivatives of the PPARα/γ dual agonist 1 allowed us to identify the ligand ( S)-6 as a potent partial agonist of both PPARα and γ subtypes. X-ray studies in PPARγ revealed two different binding modes of ( S)-6 to the canonical site. However, ( S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPARγ antagonist and supported from docking experiments. This compound did not activate the PPARγ-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, ( S)-6 inhibited the Cdk5-mediated phosphorylation of PPARγ at serine 273 that is currently considered the mechanism by which some PPARγ partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPARα/γ dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.

Cyclin-dependent kinase 5 (CDK5) is an unusual CDK whose function has been implicated in protecting the central nervous system (CNS) from oxidative damage. However, there have been few studies of CDK5 in insects. In this study, we identified the AccCDK5 gene from Apis cerana cerana and investigated its role in oxidation resistance. We found that AccCDK5 is highly conserved across species and contains conserved features of the CDK5 family. The results of qPCR analysis indicated that AccCDK5 is highly expressed during the larval and pupal stages and in the adult head and muscle. We further observed that AccCDK5 is induced by several environmental oxidative stresses. Moreover, the overexpression of the AccCDK5 protein in E. coli enhances the resistance of the bacteria to oxidative stress. The activation of CDK5 requires binding to its activator. Therefore, we also identified and cloned cyclin-dependent kinase 5 regulatory subunit 1, which we named AccCDK5r1, from Apis cerana cerana. AccCDK5r1 contains a conserved cell localization targeting domain as well as binding and activation sites for CDK5. Yeast two-hybrid analysis demonstrated the interaction between AccCDK5 and AccCDK5r1. The expression patterns of the two genes were similar after stress treatment. Collectively, these results suggest that AccCDK5 plays a pivotal role in the response to oxidative stresses and that AccCDK5r1 is a potential activator of AccCDK5.

Novel 4,5-quinolimide-based fluorophores are more solvatochromic and red-shifted than known naphthalimide analogues. Conjugation of one of these fluorophores to a peptide derived from CDK5 kinase demonstrated its sensitivity for monitoring the interaction with its regulatory partner p25. Introduction of the quinolimide-labelled peptide into living glioblastoma cells probed the interaction with endogenous p25.

Roscovitine derivatives are potent inhibitors of cyclin-dependent kinase 5 (CDK5), but they exhibit different activities, which has not been understood clearly up to now. On the other hand, the task of drug design is difficult because of the fuzzy binding mechanism. In this context, the methods of molecular docking, molecular dynamics (MD) simulation, and binding free energy analysis are applied to investigate and reveal the detailed binding mechanism of four roscovitine derivatives with CDK5. The electrostatic and van der Waals interactions of the four inhibitors with CDK5 are analyzed and discussed. The calculated binding free energies in terms of MM-PBSA method are consistent with experimental ranking of inhibitor effectiveness for the four inhibitors. The hydrogen bonds of the inhibitors with Cys83 and Lys33 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the pivotal contacts with Ile10 and Leu133 have larger contributions to the binding free energy and play critical roles in distinguishing the variant bioactivity of four inhibitors. In terms of binding mechanism of the four inhibitors with CDK5 and energy contribution of fragments of each inhibitor, two new CDK5 inhibitors are designed and have stronger inhibitory potency., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP's E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIP(S20A), but not CHIP(WT), attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli.

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that plays a major role in the regulation of glucose and lipid metabolisms and has, therefore, many implications in modern-life metabolic disorders such as diabetes, obesity, and cardiovascular diseases. Phosphorylation of PPARγ by the cyclin-dependent kinase 5 (Cdk5) has been recently proved to promote obesity and loss of insulin sensitivity. The inhibition of this reaction is currently being pursued to develop PPARγ ligands for type 2 diabetes treatments. The knowledge of the protein-protein interactions between Cdk5/p25 and PPARγ can be an important asset for better understanding of the molecular basis of the Cdk5-meditated phosphorylation of PPARγ and its inhibition. By means of a computational approach that combines protein-protein docking and adaptive biasing force molecular dynamics simulations, we obtained PPARγ-Cdk5/p25 structural models that are consistent with the mechanism of the enzymatic reaction and with overall structural features of the full length PPARγ-RXRα heterodimer bound to DNA. In addition to the active site, our model shows that the interacting regions between the two proteins should involve two distal docking sites, comprising the PPARγ Ω-loop and Cdk5 N-terminal lobe and the PPARγ β-sheet and Cdk5 C-terminal lobe. These sites are related to PPARγ transactivation and directly interact with PPARγ ligands. Our results suggest that β-sheets and Ω-loop stabilization promoted by PPARγ agonists could be important to inhibit Cdk5-mediated phosphorylation.

A method is proposed to study protein-ligand binding in a system governed by specific and nonspecific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultraweak associations lead instead to broader distributions, a manifestation of nonspecific, sparsely populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (prerelaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated., (© 2015 Wiley Periodicals, Inc.)

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Development of multi-target drugs is becoming increasingly attractive in the repertoire of protein kinase inhibitors discovery. In this study, we carried out molecular docking, molecular dynamics simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations, principal component analysis (PCA), and dynamical cross-correlation matrices (DCCM) to dissect the molecular mechanism for the valmerin-19 acting as a dual inhibitor for glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5). Detailed MM-PBSA calculations revealed that the binding free energies of the valmerin-19 to GSK3β/CDK5 were calculated to be -12.60 ± 2.28 kcal mol(-1) and -11.85 ± 2.54 kcal mol(-1), respectively, indicating that valmerin-19 has the potential to act as a dual inhibitor of GSK3β/CDK5. The analyses of PCA and DCCM results unraveled that binding of the valmerin-19 reduced the conformational dynamics of GSK3β/CDK5 and the valmerin-19 bound to GSK3β/CDK5 might occur mostly through a conformational selection mechanism. This study may be helpful for the future design of novel and potent dual GSK3β/CDK5 inhibitors.

In this study, we applied steered molecular dynamics (SMD) simulations to investigate the unbinding mechanism of nine inhibitors of the enzyme cyclin-dependent kinase 5 (CDK5). The study had two major objectives: (i) to create a correlation between the unbinding force profiles and the inhibition activities of these compounds expressed as IC50 values; (ii) to investigate the unbinding mechanism and to reveal atomistic insights, which could help identify accessory binding sites and transient interactions. Overall, we carried out 1.35 μs of cumulative SMD simulations. We showed that SMD could qualitatively discriminate binders from nonbinders, while it failed to properly rank series of inhibitors, particularly when IC50 values were too similar. From a mechanistic standpoint, SMD provided useful insights related to transient and dynamical interactions, which could complement static description obtained by X-ray crystallography experiments. In conclusion, the present study represents a further step toward a systematic exploitation of SMD and other dynamical approaches in structure-based drug design and computational medicinal chemistry.

Multiple lines of evidence link the incidence of diabetes to the development of Alzheimer's disease (AD). Patients with diabetes have a 50 to 75% increased risk of developing AD. Cyclin dependent kinase 5 (Cdk5) is a serine/threonine protein kinase, which forms active complexes with p35 or p39, found principally in neurons and in pancreatic β cells. Recent studies suggest that Cdk5 hyperactivity is a possible link between neuropathology seen in AD and diabetes. Previously, we identified P5, a truncated 24-aa peptide derived from the Cdk5 activator p35, later modified as TFP5, so as to penetrate the blood-brain barrier after intraperitoneal injections in AD model mice. This treatment inhibited abnormal Cdk5 hyperactivity and significantly rescued AD pathology in these mice. The present study explores the potential of TFP5 peptide to rescue high glucose (HG)-mediated toxicity in rat embryonic cortical neurons. HG exposure leads to Cdk5-p25 hyperactivity and oxidative stress marked by increased reactive oxygen species production, and decreased glutathione levels and superoxide dismutase activity. It also induces hyperphosphorylation of tau, neuroinflammation as evident from the increased expression of inflammatory cytokines like TNF-α, IL-1β, and IL-6, and apoptosis. Pretreatment of cortical neurons with TFP5 before HG exposure inhibited Cdk5-p25 hyperactivity and significantly attenuated oxidative stress by decreasing reactive oxygen species levels, while increasing superoxide dismutase activity and glutathione. Tau hyperphosphorylation, inflammation, and apoptosis induced by HG were also considerably reduced by pretreatment with TFP5. These results suggest that TFP5 peptide may be a novel candidate for type 2 diabetes therapy.

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.

Cyclin-dependent kinases (CDKs) belong to the CMGC subfamily of protein kinases and play crucial roles in eukaryotic cell division cycle. At least seven different CDKs have been reported to be implicated in the cell cycle regulation in vertebrates. These CDKs are highly homologous and contain a conserved catalytic core. This makes the design of inhibitors specific for a particular CDK difficult. There is, however, growing need for CDK5 specific inhibitors to treat various neurodegenerative diseases. Recently, cis-substituted cyclobutyl-4-aminoimidazole inhibitors have been identified as potent CDK5 inhibitors that gave up to 30-fold selectivity over CDK2. Available IC50 values also indicate a higher potency of this class of inhibitors over commercially available drugs, such as roscovitine. To understand the molecular basis of higher potency and selectivity of these inhibitors, here, we present molecular dynamics simulation results of CDK5/p25 and CDK2/CyclinE complexed with a series of cyclobutyl-substituted imidazole inhibitors and roscovitine. The atomic details of the stereospecificity and selectivity of these inhibitors are obtained from energetics and binding characteristics to the CDK binding pocket. The study not only complements the experimental findings, but also provides a wealth of detailed information that could help the structure-based drug designing processes.

Molecular docking, molecular dynamics (MD) simulations, and binding free energy analysis were performed to reveal differences in the binding affinities between five 2-aminothiazole inhibitors and CDK5. The hydrogen bonding and hydrophobic interactions between inhibitors and adjacent residues are analyzed and discussed. The rank of calculated binding free energies using the MM-PBSA method is consistent with experimental result. The results illustrate that hydrogen bonds with Cys83 favor inhibitor binding. The van der Waals interactions, especially the important contact with Ile10, dominate in the binding free energy and play a crucial role in distinguishing the different bioactivity of the five inhibitors.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 ((132)LLINRKGELKLADFGLARAFGIP(154)) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The microtubule (MT)-associated protein tau attaches to neuronal MT networks and regulates their integrity. The phosphorylation state of tau alters its binding activity. MT integrity is maintained by the phosphorylation state of tau, which is under the control of the kinase-phosphatase balance. This control requires the proper regulation of topographical and temporal characteristics of tau kinases and phosphatases. The tau phosphorylation protein complex (TPPC) anchors tau kinases and phosphatases via scaffold proteins, tau effectors, and tau itself. Targeting these proteins in TPPC fulfills the topographical requirements for maintaining MT functions. The switching of tau kinase activity determines the order of the kinase action. The combined action of kinases is temporally modulated; reversal of the time order of events results in a differential state of tau phosphorylation. Elucidation of protein-protein interaction in the regulation of tau phosphorylation will shed light on the physiology and pathology of tau phosphorylation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A series of novel 1-acyl-3-amino-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives were designed and synthesized. These derivatives were initially evaluated for their in vitro anticancer activity against human colon carcinoma HCT-116 cell line, and compounds 11a, b were chosen for further evaluation their in vitro activity against other five human cancer cell lines. These results indicate that most of the target compounds have considerable in vitro anticancer activity. The most active compound 11a was found to be 4- to 28-fold more potent than (R)-roscovitine against six human cancer cell lines. In addition, compound 11a was assessed for its activity against 12 kinases, and then evaluated for its interaction mode by docking experiments with cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase-3β (GSK3β)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles was synthesized through a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3Kα and showed promising inhibitory activities against PI3Kα with most IC(50) values in the micromolar range. Among them, compound 18 was strongly considered as the most interesting compound with an IC(50) value of 0.091 μM. This series exhibited also significant anti-proliferative effects in various human cancer cell lines including those resulting in activation of the PI3K pathway., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Hu antigen R (HuR) is an mRNA-binding protein belonging to the ELAV family. It is highly expressed in cancer and involved in cell survival and proliferation. The impact of post-translational regulation of HuR and resulting cellular effects are poorly understood. In the current report, we describe a direct interaction between HuR and Cdk5 in glioma. We determined that Cdk5 specifically phosphorylates HuR at the serine 202 residue in the unique hinge region. The molecular consequences of this interaction are an altered HuR ability to bind, stabilize, and promote translation of mRNAs. At the cellular level, the anomalous HuR phosphorylation at this site evokes robust defects in centrosome duplication and cohesion as well as arrest of cell cycle progression. Subcellular fractionation and immunofluorescence technique confirm a direct integration of HuR and Cdk5 with centrosomes. We propose that HuR stores mRNA in the centrosome and that HuR phosphorylation by Cdk5 controls de novo protein synthesis in near proximity to centrosomes and, thus, impacts centrosome function.

Large-scale conformational transitions represent both a challenge and an opportunity for computational drug design. Exploring the conformational space of a druggable target with sufficient detail is computationally demanding. However, if it were possible to fully account for target flexibility, one could exploit this knowledge to rationally design more potent and more selective drug candidates. Here, we discuss how molecular dynamics together with free energy algorithms based on Metadynamics and Path Collective Variables can be used to study both large-scale conformational transitions and ligand binding to flexible targets. We show real-life examples of how these methods have been applied in the case of cyclin-dependent kinases, a family of flexible targets that shows promise in cancer therapy.

The present study reports the development of a template for the active binding site of Cdk5 for structure-based drug design. The developed template of Cdk5 was validated by redocking with ligands I (PBD code 1UNG), II (PBD code 1UNL) and III (PBD code 1UNH). The results demonstrate a good match of the docked and the crystallographic binding orientations with RMSD less than 2.0Å. The validation results show that the constructed Cdk5 template is a good model system for predicting ligand binding orientations and binding affinities. Furthermore, the developed template was applied to predict binding mode and binding affinity of thirty-six known Cdk5 inhibitors. The results showed that the binding energy of almost Cdk5 inhibitors related to their biological evaluation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.

The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The lack of selectivity of all existing ATP competitive inhibitors for a single cyclin-dependent kinase (CDK) has led us to redirect the structure-based molecule design from targeting the classic ATP-binding pocket in CDK5 toward the CDK5/p25 interface. The aim was to seek novel inhibition mechanisms to interrupt protein-protein interactions. A combined strategy of alanine-scanning calculations for locating binding sites, virtual screening for small molecules, molecular dynamics simulations for examining the binding stability of virtual screening hits and bio-assays for testing the level of inhibition was set up and used to explore novel inhibitors capable of interrupting the interactions between the proteins, and consequently of inhibiting the kinase activity. Two compounds were shown to inhibit the complex formation between CDK5 and p25 through p25 binding. They could open avenues for the discovery of new types of structures that prevent interactions between CDK5 and p25 or other CDK and activator proteins, and, more importantly, provide leads in the development of selective inhibitors among CDKs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Structure-based 3D-QSAR approaches (CoMFA and CoMSIA) were applied to understand the structural requirements of the Cyclin-dependent kinase 5/p25 inhibitors. Cyclin-dependent kinase 5 (CDK5) is believed to play an important role in the development of the central nervous system during the process of mammalian embryogenesis. Genetic algorithm based docking program (GOLD) was successfully utilized to orient the compounds inside the binding pocket of the CDK5/p25 structure. The adapted alignment method with the suitable parameters resulted in a reliable model. Furthermore, the final model was robust enough to forecast the activities of test compounds, satisfactorily. The contour maps were produced around the functional groups to understand the SAR requirements. Moreover, we also investigate the structural attributes of the inhibitors which make them selective toward CDK5/p25 over its close counterpart, i.e., CDK2. The study could be helpful to rationalize the new compounds with better inhibition and selectivity profiles against CDK5/p25.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is increasingly implicated in various neurodegenerative diseases. Deregulated Cdk5 activity has been associated with neuronal death, but the underlying mechanisms are not well understood. Here we report an unexpected role for Cdk5 in the regulation of induced autophagy in neurons. We have identified endophilin B1 (EndoB1) as a Cdk5 substrate, and show that Cdk5-mediated phosphorylation of EndoB1 is required for autophagy induction in starved neurons. Furthermore, phosphorylation of EndoB1 facilitates EndoB1 dimerization and recruitment of UVRAG (UV radiation resistance-associated gene). More importantly, Cdk5-mediated phosphorylation of EndoB1 is essential for autophagy induction and neuronal loss in models of Parkinson's disease. Our findings not only establish Cdk5 as a critical regulator of autophagy induction, but also reveal a role for Cdk5 and EndoB1 in the pathophysiology of Parkinson's disease through modulating autophagy.

High mobility group box 1 (HMGB1) protein is the most abundant chromatin-associated non-histone protein expressed in all nucleated eukaryotic cells. We examined the phosphorylation of mammalian HMGB1 by testing the ability of the cyclin-dependent kinase 5 (Cdk5) to use as substrates native protein, either unmodified or in vivo acetylated and recombinant HMGB1. It turned out that Cdk5 was active on the in vivo acetylated HMGB1 only. We studied the effect of the phosphorylation on the 'architectural' properties of the acetylated HMGB1. The treatment with Cdk5 of the acetylated HMGB1 inhibited its capacity to induce DNA end-joining but had no effect on its ability to recognize distorted DNA structures.

Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a β-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that β-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys(245)-Ala(277). p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1-42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

The crystal structure of the cdk5/p25 complex has provided information on possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase. Comparative molecular dynamics simulations are reported here for physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5., (Published by Elsevier Ltd.)

Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimer's disease-associated kinases that promote the formation of hyperphosphorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimer's disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg(2+) in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in k(cat) and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg(2+) does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg(2+) as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 +/- 0.1 in the presence of the second Mg(2+) was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. The pH profile revealed a residue with a pK(a) of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer.

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein-protein recognition characteristics between the kinase and its activator.

The cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases, playing an essential role in regulating cell-cycle progression. In our present work, human CDK5 and a novel CDK5 splicing variant, named as CDK5-SV, were cloned from the cDNA library of human testis. CDK5-SV lacking the exon 7 of CDK5 encodes a protein of 260 amino acids. Through RT-PCR analysis in different human tissues, CDK5-SV was found to be expressed in testis, skeletal muscle, colon, bone marrow and ovary, while CDK5 was ubiquitously expressed. Immunofluorescence experiment in HeLa cells showed that the subcellular localizations of CDK5-SV and CDK5 were totally different. CDK5 mainly located in the cytoplasm, while CDK5-SV accumulated in nucleus. Reporter gene assay showed that when co-transfected with beta-catenin, CDK5 and CDK5-SV could both strongly inhibit the Wnt/beta-catenin signaling pathway. Consistently, CDK5-SV could also interact with beta-catenin as CDK5 does. Taken together, our findings suggest that CDK5-SV might also be a negative regulator of Wnt/beta-catenin signaling pathway.

While most cyclin-dependent kinases (CDKs) are involved in cell cycle control, CDK5 is mostly known for crucial functions in neurogenesis. However, we cloned sea urchin CDK5 from a two-cell stage cDNA library and found that the protein is present in eggs and embryos, up to the pluteus stage, but without associated kinase activity. To investigate the potential for nonneuronal roles, we screened a starfish cDNA library with the yeast two-hybrid system, for possible CDK5 partners. Interactions with clones expressing part of cyclin B3 and cyclin E proteins were found and the full-length cyclins were cloned. These interactions were verified in vitro but not in extracts of starfish oocytes and embryos, at any stages, despite the presence of detectable amounts of CDK5, cyclin B3, and cyclin E. We then looked for p35, the CDK5-specific activator, and cloned the sea urchin ortholog. A sea urchin-specific anomaly in the amino acid sequence is the absence of N-terminal myristoylation signal, but nucleotide environment analysis suggests a much higher probability of translation initiation on the second methionine(Met44), that is associated with a conserved myristoylation signal. p35 was found to associate with CDK5 and, when bacterially produced, to confer protein kinase activity to CDK5 immunoprecipitated from sea urchin eggs and embryos. However, p35 mRNA expression was found to begin only at the end of the blastula stage, and the protein was undetectable at any embryonic stage, suggesting a neuronal role beginning in late larval stages.

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.