SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3a and GSK3β are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3β, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3β is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity

Molecular insights into conformational dynamics associated with the open-closed-phosphorylated states of PITPα

AbstractPhosphatidylinositol transfer proteins (PITPα) are lipid carrier proteins that are involved in replenishing the phosphatidylinositol lipid molecules on the plasma membranes. Transitions between the open and closed state conformations are necessary steps in the mechanism of lipid transfer by PITPα. The apo (open state) conformation is assumed to occur at the membrane surface during the lipid exchange while the lipid bound (closed state) conformation is required for transfer activity. The transfer of phosphatidylinositol by PITPα is controlled by the phosphorylation of S166 in its regulatory region. We wanted to decipher the molecular basis for structure-function relationship between the open-closed-phosphorylated states of PITPα. We used all-atom Molecular Dynamics Simulations to study the conformational dynamics in each of these states. Our study shows that the open state is highly dynamic and its transition to closed state would stabilize PITPα. We observed restricted conformational sampling of the phosphorylated state which provide basis for its decreased lipid transfer activity. Further, using analysis of residue-residue contact maps and hydrogen bond interactions we discuss the impact of phosphorylation on the global conformation of PITPα. Overall, our work provides insights into the structural dynamics in each state and their functional significance.</jats:p

Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A

RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg²⁺ are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants

Atypical switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica

Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Mutations that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine

2.09 Å Resolution structure of <i>E. coli</i> HigBA toxin–antitoxin complex reveals an ordered DNA-binding domain and intrinsic dynamics in antitoxin

The toxin–antitoxin (TA) systems are small operon systems that are involved in important physiological processes in bacteria such as stress response and persister cell formation. Escherichia coli HigBA complex belongs to the type II TA systems and consists of a protein toxin called HigB and a protein antitoxin called HigA. The toxin HigB is a ribosome-dependent endoribonuclease that cleaves the translating mRNAs at the ribosome A site. The antitoxin HigA directly binds the toxin HigB, rendering the HigBA complex catalytically inactive. The existing biochemical and structural studies had revealed that the HigBA complex forms a heterotetrameric assembly via dimerization of HigA antitoxin. Here, we report a high-resolution crystal structure of E. coli HigBA complex that revealed a well-ordered DNA binding domain in HigA antitoxin. Using SEC-MALS and ITC methods, we have determined the stoichiometry of complex formation between HigBA and a 33 bp DNA and report that HigBA complex as well as HigA homodimer bind to the palindromic DNA sequence with nano molar affinity. Using E. coli growth assays, we have probed the roles of key, putative active site residues in HigB. Spectroscopic methods (CD and NMR) and molecular dynamics simulations study revealed intrinsic dynamic in antitoxin in HigBA complex, which may explain the large conformational changes in HigA homodimer in free and HigBA complexes observed previously. We also report a truncated, heterodimeric form of HigBA complex that revealed possible cleavage sites in HigBA complex, which can have implications for its cellular functions.</jats:p

SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3 alpha and GSK3 beta are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3 beta, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3 beta is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity

SIRT2 regulates oxidative stress-induced cell death through deacetylation of c-Jun NH2-terminal kinase

c-Jun NH2-terminal kinases (JNKs) are responsive to stress stimuli and their activation regulate key cellular functions, including cell survival, growth, differentiation and aging. Previous studies demonstrate that activation of JNK requires dual phosphorylation by the mitogen-activated protein kinase kinases. However, other post-translational mechanisms involved in regulating the activity of JNK have been poorly understood. In this work, we studied the functional significance of reversible lysine acetylation in regulating the kinase activity of JNK. We found that the acetyl transferase p300 binds to, acetylates and inhibits kinase activity of JNK. Using tandem mass spectrometry, molecular modelling and molecular dynamics simulations, we found that acetylation of JNK at Lys153 would hinder the stable interactions of the negatively charged phosphates and prevent the adenosine binding to JNK. Our screening for the deacetylases found SIRT2 as a deacetylase for JNK. Mechanistically, SIRT2-dependent deacetylation enhances ATP binding and enzymatic activity of JNK towards c-Jun. Furthermore, SIRT2-mediated deacetylation favours the phosphorylation of JNK by MKK4, an upstream kinase. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. Conversely, SIRT2 inhibition attenuates H2O2-mediated cell death in HeLa cells. SIRT2-deficient (SIRT2-KO) mice exhibit increased acetylation of JNK, which is associated with markedly reduced catalytic activity of JNK in the liver. Interestingly, SIRT2-KO mice were resistant to acetaminophen-induced liver toxicity. SIRT2-KO mice show lower cell death, minimal degenerative changes, improved liver function and survival following acetaminophen treatment. Overall, our work identifies SIRT2-mediated deacetylation of JNK as a critical regulator of cell survival during oxidative stress