Metabolic and circadian inputs encode anticipatory biogenesis of hepatic fed microRNAs.

Starvation and refeeding are mostly unanticipated in the wild in terms of duration, frequency, and nutritional value of the refed state. Notwithstanding this, organisms mount efficient and reproducible responses to restore metabolic homeostasis. Hence, it is intuitive to invoke expectant molecular mechanisms that build anticipatory responses to enable physiological toggling during fed-fast cycles. In this regard, we report anticipatory biogenesis of oscillatory hepatic microRNAs that peak during a fed state and inhibit starvation-responsive genes. Our results clearly demonstrate that the levels of primary and precursor microRNA transcripts increase during a fasting state, in anticipation of a fed response. We delineate the importance of both metabolic and circadian cues in orchestrating hepatic fed microRNA homeostasis in a physiological setting. Besides illustrating metabo-endocrine control, our findings provide a mechanistic basis for the overarching influence of starvation on anticipatory biogenesis. Importantly, by using pharmacological agents that are widely used in clinics, we point out the high potential of interventions to restore homeostasis of hepatic microRNAs, whose deregulated expression is otherwise well established to cause metabolic diseases.

Improving In Vivo Tumor Accumulation and Efficacy of Platinum Antitumor Agents by Electronic Tuning of the Kinetic Lability.

The impact of kinetic lability or reactivity on in vitro cytotoxicity, stability in plasma, in vivo tumor and tissue accumulation, and antitumor efficacy of functional platinum(II) (Pt) anticancer agents containing a O˄O ß-diketonate leaving ligand remain largely unexplored. To investigate this, we synthesized Pt complexes [(NH3 )2 Pt(L1-H)]NO3 and [(DACH)Pt(L1-H)]NO3 (L1=4,4,4-trifluoro-1-ferrocenylbutane-1,3-dione, DACH=1R,2R-cyclohexane-1,2-diamine) containing an electron deficient [L1-H]- O˄O leaving ligand and [(NH3 )2 Pt(L2-H)]NO3 and [(DACH)Pt(L2-H)]NO3 (L2=1-ferrocenylbutane-1,3-dione) containing an electron-rich [L2-H]- O˄O leaving ligand. While all four complexes have comparable lipophilicity, the presence of the electron-withdrawing CF3 group was found to dramatically enhance the reactivity of these complexes toward nucleophilic biomolecules. In vitro cellular assays revealed that the more reactive complexes have higher cellular uptake and higher anticancer potency as compared to their less reactive analogs. But the scenario is opposite in vivo, where the less reactive complex showed improved tissue and tumor accumulation and better anticancer efficacy in mice bearing ovarian xenograft when compared to its more reactive analog. Finally, in addition to demonstrating the profound but contrasting impact of kinetic lability on in vitro and in vivo antitumor potencies, we also described the impact of kinetic lability on the mechanism of action of this class of promising antitumor agents.

Metabolic transitions regulate global protein fatty acylation.

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival.

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that the coordinated action of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) orchestrate cholesterol build-up in in vitro and in vivo mouse models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while SIRT6 along with G9a represses the genes involved in cholesterol efflux. The accumulated cholesterol in Mtb infected macrophages promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and pharmacological inhibition in vivo; or utilization of BMDMs derived from Sirt6-/- mice or in vivo infection in haplo-insufficient Sirt6-/+ mice; hampered host cholesterol accumulation and restricted Mtb burden. These findings shed light on the novel roles of G9a and SIRT6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

Shaping mitochondria through fed-fast and circadian cycles.

Energy and metabolic homeostasis at the level of the whole body are dictated by the balance between nutrient intake/utilization, bioenergetic potential, and energy expenditure, which are tightly coupled with fed/fast cycles and circadian oscillation. Emerging literature has highlighted the importance of each of these mechanisms that are essential to maintain physiological homeostasis. Lifestyle changes predominantly associated with altered fed-fast and circadian cycles are well established to affect systemic metabolism and energetics, and hence contribute to pathophysiological states. Therefore, it is not surprising that mitochondria have emerged as being pivotal in maintaining physiological homeostasis through daily oscillations/fluctuations in nutrient inputs and light-dark/sleep-wake cycles. Moreover, given the inherent association between mitochondrial dynamics/morphology and functions, it is important to understand the phenomenological and mechanistic underpinnings of fed-fast and circadian cycles dependent remodeling of mitochondria. In this regard, we have summarized the current status of the field in addition to providing a perspective vis-a-vis the complexity of cell-autonomous and non-cell-autonomous signals that dictate mitochondrial dynamics. We also highlight the lacunae besides speculating on prospective efforts that will possibly redefine our insights into the diurnal orchestration of fission/fusion events, which are ultimately coupled to the mitochondrial output.

Metabolic regulation of CTCF expression and chromatin association dictates starvation response in mice and flies.

Coordinated temporal control of gene expression is essential for physiological homeostasis, especially during metabolic transitions. However, the interplay between chromatin architectural proteins and metabolism in regulating transcription is less understood. Here, we demonstrate a conserved bidirectional interplay between CTCF (CCCTC-binding factor) expression/function and metabolic inputs during feed-fast cycles. Our results indicate that its loci-specific functional diversity is associated with physiological plasticity in mouse hepatocytes. CTCF differential expression and long non-coding RNA-Jpx mediated changes in chromatin occupancy, unraveled its paradoxical yet tuneable functions, which are governed by metabolic inputs. We illustrate the key role of CTCF in controlling temporal cascade of transcriptional response, with effects on hepatic mitochondrial energetics and lipidome. Underscoring the evolutionary conservation of CTCF-dependent metabolic homeostasis, CTCF knockdown in flies abrogated starvation resistance. In summary, we demonstrate the interplay between CTCF and metabolic inputs that highlights the coupled plasticity of physiological responses and chromatin function.

A Rationally Designed Bimetallic Platinum (II)-Ferrocene Antitumor Agent Induces Non-Apoptotic Cell Death and Exerts in Vivo Efficacy.

Despite phenomenal clinical success, the efficacy of platinum anticancer drugs is often compromised due to inherent and acquired drug resistant phenotypes in cancers. To circumvent this issue, we designed two heterobimetallic platinum (II)-ferrocene hybrids that display multi-pronged anticancer action. In cancer cells, our best compound, 2, platinates DNA, produces reactive oxygen species, and has nucleus, mitochondria, and endoplasmic reticulum as potential targets. The multi-modal mechanism of action of these hybrid agents lead to non-apoptotic cell death induction which enables circumventing apoptosis resistance and significant improvement in platinum cross resistance profile. Finally, in addition to describing detail mechanistic insights, we also assessed its stability in plasma and demonstrate anticancer efficacy in an in vivo A2780 xenograft model. Strikingly, compared to oxaliplatin, our compound displays better tolerability, safety profile and efficacy in vivo.

Drosophila Sirtuin 6 mediates developmental diet-dependent programming of adult physiology and survival.

Organisms in the wild experience unpredictable and diverse food availability throughout their lifespan. Over-/under-nutrition during development and in adulthood is known to dictate organismal survival and fitness. Studies using model systems have also established long-term effects of developmental dietary alterations on life-history traits. However, the underlining genetic/molecular factors, which differentially couple nutrient inputs during development with fitness later in life are far less understood. Using Drosophila and loss/gain of function perturbations, our serendipitous findings demonstrate an essential role of Sirtuin 6 in regulating larval developmental kinetics, in a nutrient-dependent manner. The absence of Sirt6 affected ecdysone and insulin signalling and led to accelerated larval development. Moreover, varying dietary glucose and yeast during larval stages resulted in enhanced susceptibility to metabolic and oxidative stress in adults. We also demonstrate an evolutionarily conserved role for Sirt6 in regulating physiological homeostasis, physical activity and organismal lifespan, known only in mammals until now. Our results highlight gene-diet interactions that dictate thresholding of nutrient inputs and physiological plasticity, operative across development and adulthood. In summary, besides showing its role in invertebrate ageing, our study also identifies Sirt6 as a key factor that programs macronutrient-dependent life-history traits.

Continuous variable responses and signal gating form kinetic bases for pulsatile insulin signaling and emergence of resistance.

Understanding kinetic control of biological processes is as important as identifying components that constitute pathways. Insulin signaling is central for almost all metazoans, and its perturbations are associated with various developmental disorders, metabolic diseases, and aging. While temporal phosphorylation changes and kinetic constants have provided some insights, constant or variable parameters that establish and maintain signal topology are poorly understood. Here, we report kinetic parameters that encode insulin concentration and nutrient-dependent flow of information using iterative experimental and mathematical simulation-based approaches. Our results illustrate how dynamics of distinct phosphorylation events collectively contribute to selective kinetic gating of signals and maximum connectivity of the signaling cascade under normo-insulinemic but not hyper-insulinemic states. In addition to identifying parameters that provide predictive value for maintaining the balance between metabolic and growth-factor arms, we posit a kinetic basis for the emergence of insulin resistance. Given that pulsatile insulin secretion during a fasted state precedes a fed response, our findings reveal rewiring of insulin signaling akin to memory and anticipation, which was hitherto unknown. Striking disparate temporal behavior of key phosphorylation events that destroy the topology under hyper-insulinemic states underscores the importance of unraveling regulatory components that act as bandwidth filters. In conclusion, besides providing fundamental insights, our study will help in identifying therapeutic strategies that conserve coupling between metabolic and growth-factor arms, which is lost in diseases and conditions of hyper-insulinemia.

Intervention by picroside II on FFAs induced lipid accumulation and lipotoxicity in HepG2 cells.

BACKGROUND: Accumulation of free fatty acids (FFAs) in hepatocytes is a hallmark of liver dysfunction and non-alcoholic fatty liver disease (NAFLD). Excessive deposition of FFAs alters lipid metabolism pathways increasing the oxidative stress and mitochondrial dysfunction. Attenuating hepatic lipid accumulation, oxidative stress, and improving mitochondrial function could provide potential targets in preventing progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). Earlier studies with Picrorhiza kurroa extract have shown reduction in hepatic damage and fatty acid infiltration in several experimental models and also clinically in viral hepatitis. Thus, the effect of P. kurroa's phytoactive, picroside II, needed mechanistic investigation in appropriate in vitro liver cell model. OBJECTIVE(S): To study the effect of picroside II on FFAs accumulation, oxidative stress and mitochondrial function with silibinin as a positive control in in vitro NAFLD model. MATERIALS AND METHODS: HepG2 cells were incubated with FFAs-1000µM in presence and absence of Picroside II-10 µM for 20 hours. RESULTS: HepG2 cells incubated with FFAs-1000µM lead to increased lipid accumulation. Picroside II-10µM attenuated FFAs-induced lipid accumulation (33%), loss of mitochondrial membrane potential (ΔΨm), ATP depletion, and production of reactive oxygen species (ROS). A concomitant increase in cytochrome C at transcription and protein levels was observed. An increase in expression of MnSOD, catalase, and higher levels of tGSH and GSH:GSSG ratios underlie the ROS salvaging activity of picroside II. CONCLUSION: Picroside II significantly attenuated FFAs-induced-lipotoxicity. The reduction in ROS, increased antioxidant enzymes, and improvement in mitochondrial function underlie the mechanisms of action of picroside II. These findings suggest a need to develop an investigational drug profile of picroside II for NAFLD as a therapeutic strategy. This could be evaluated through the fast-track path of reverse pharmacology.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

Dendritic Fibrous Nanosilica (DFNS) for RNA Extraction from Cells.

Efficient RNA extraction is critical for all downstream molecular applications and techniques. Despite the availability of several commercial kits, there is an enormous scope to develop novel materials that have high binding and elution capacities. Here, we show that RNA from the cells can be extracted by dendritic fibrous nanosilica (DFNS) with higher efficiency than commercially available silicas. This could be because of the unique fibrous morphology, high accessible surface area, and nanosize particles of DFNS. We studied various fundamental aspects, including the role of particle size, morphology, surface area, and charge on the silica surface in RNA extraction efficiency. Fourier transform infrared (FTIR) spectroscopy studies revealed the interaction of functional groups of RNA with the silica surface, causing selective binding. Due to the sustainable synthesis protocol of DFNS and the simplicity of various buffers and washing solutions used, this RNA extraction kit can be assembled in any lab. In addition to the fundamental aspects of DFNS-RNA interactions, this study has the potential to initiate the development of indigenous DFNS-based kits for RNA extraction.

Search and Capture: Disorder Rules Gene Promoter Selection.

Intrinsically disordered regions (IDRs) are preponderant in transcription factors (TFs) and are evolutionarily less conserved vis-à-vis DNA-binding domains (DBDs). Unexpected findings from Barkai and colleagues, which demonstrate that promoter selectivity is determined by IDRs, should significantly enhance our understanding of gene expression regulation.

Spatiotemporal gating of SIRT1 functions by O-GlcNAcylation is essential for liver metabolic switching and prevents hyperglycemia.

Inefficient physiological transitions are known to cause metabolic disorders. Therefore, investigating mechanisms that constitute molecular switches in a central metabolic organ like the liver becomes crucial. Specifically, upstream mechanisms that control temporal engagement of transcription factors, which are essential to mediate physiological fed-fast-refed transitions are less understood. SIRT1, a NAD+-dependent deacetylase, is pivotal in regulating hepatic gene expression and has emerged as a key therapeutic target. Despite this, if/how nutrient inputs regulate SIRT1 interactions, stability, and therefore downstream functions are still unknown. Here, we establish nutrient-dependent O-GlcNAcylation of SIRT1, within its N-terminal domain, as a crucial determinant of hepatic functions. Our findings demonstrate that during a fasted-to-refed transition, glycosylation of SIRT1 modulates its interactions with various transcription factors and a nodal cytosolic kinase involved in insulin signaling. Moreover, sustained glycosylation in the fed state causes nuclear exclusion and cytosolic ubiquitin-mediated degradation of SIRT1. This mechanism exerts spatiotemporal control over SIRT1 functions by constituting a previously unknown molecular relay. Of note, loss of SIRT1 glycosylation discomposed these interactions resulting in aberrant gene expression, mitochondrial dysfunctions, and enhanced hepatic gluconeogenesis. Expression of nonglycosylatable SIRT1 in the liver abrogated metabolic flexibility, resulting in systemic insulin resistance, hyperglycemia, and hepatic inflammation, highlighting the physiological costs associated with its overactivation. Conversely, our study also reveals that hyperglycosylation of SIRT1 is associated with aging and high-fat-induced obesity. Thus, we establish that nutrient-dependent glycosylation of SIRT1 is essential to gate its functions and maintain physiological fitness.

Metabolic choreography of gene expression: nutrient transactions with the epigenome.

Eukaryotic complexity and thus their ability to respond to diverse cues are largely driven by varying expression of gene products, qualitatively and quantitatively. Protein adducts in the form of post-translational modifications, most of which are derived from metabolic intermediates, allow fine tuning of gene expression at multiple levels. With the advent of high-throughput and high-resolution mapping technologies there has been an explosion in terms of the kind of modifications on chromatin and other factors that govern gene expression. Moreover, even the classical notion of acetylation and methylation dependent regulation of transcription is now known to be intrinsically coupled to biochemical pathways, which were otherwise regarded as 'mundane'. Here we have not only reviewed some of the recent literature but also have highlighted the dependence of gene regulatory mechanisms on metabolic inputs, both direct and indirect. We have also tried to bring forth some of the open questions, and how our understanding of gene expression has changed dramatically over the last few years, which has largely become metabolism centric. Finally, metabolic regulation of epigenome and gene expression has gained much traction due to the increased incidence of lifestyle and age-related diseases.

Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria.

Anabolic and catabolic signaling mediated via mTOR and AMPK (AMP-activated kinase) have to be intrinsically coupled to mitochondrial functions for maintaining homeostasis and mitigate cellular/organismal stress. Although glutamine is known to activate mTOR, whether and how differential mitochondrial utilization of glutamine impinges on mTOR signaling has been less explored. Mitochondrial SIRT4, which unlike other sirtuins is induced in a fed state, is known to inhibit catabolic signaling/pathways through the AMPK-PGC1α/SIRT1-peroxisome proliferator-activated receptor α (PPARα) axis and negatively regulate glutamine metabolism via the tricarboxylic acid cycle. However, physiological significance of SIRT4 functions during a fed state is still unknown. Here, we establish SIRT4 as key anabolic factor that activates TORC1 signaling and regulates lipogenesis, autophagy, and cell proliferation. Mechanistically, we demonstrate that the ability of SIRT4 to inhibit anaplerotic conversion of glutamine to α-ketoglutarate potentiates TORC1. Interestingly, we also show that mitochondrial glutamine sparing or utilization is critical for differentially regulating TORC1 under fed and fasted conditions. Moreover, we conclusively show that differential expression of SIRT4 during fed and fasted states is vital for coupling mitochondrial energetics and glutamine utilization with anabolic pathways. These significant findings also illustrate that SIRT4 integrates nutrient inputs with mitochondrial retrograde signals to maintain a balance between anabolic and catabolic pathways.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Serotonin regulates mitochondrial biogenesis and function in rodent cortical neurons via the 5-HT2A receptor and SIRT1-PGC-1α axis.

Mitochondria in neurons, in addition to their primary role in bioenergetics, also contribute to specialized functions, including regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. However, the factors that influence mitochondrial biogenesis and function in neurons remain poorly elucidated. Here, we identify an important role for serotonin (5-HT) as a regulator of mitochondrial biogenesis and function in rodent cortical neurons, via a 5-HT2A receptor-mediated recruitment of the SIRT1-PGC-1α axis, which is relevant to the neuroprotective action of 5-HT. We found that 5-HT increased mitochondrial biogenesis, reflected through enhanced mtDNA levels, mitotracker staining, and expression of mitochondrial components. This resulted in higher mitochondrial respiratory capacity, oxidative phosphorylation (OXPHOS) efficiency, and a consequential increase in cellular ATP levels. Mechanistically, the effects of 5-HT were mediated via the 5-HT2A receptor and master modulators of mitochondrial biogenesis, SIRT1 and PGC-1α. SIRT1 was required to mediate the effects of 5-HT on mitochondrial biogenesis and function in cortical neurons. In vivo studies revealed that 5-HT2A receptor stimulation increased cortical mtDNA and ATP levels in a SIRT1-dependent manner. Direct infusion of 5-HT into the neocortex and chemogenetic activation of 5-HT neurons also resulted in enhanced mitochondrial biogenesis and function in vivo. In cortical neurons, 5-HT enhanced expression of antioxidant enzymes, decreased cellular reactive oxygen species, and exhibited neuroprotection against excitotoxic and oxidative stress, an effect that required SIRT1. These findings identify 5-HT as an upstream regulator of mitochondrial biogenesis and function in cortical neurons and implicate the mitochondrial effects of 5-HT in its neuroprotective action.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.