Unstructured Conformations Are a Substrate Requirement for the Sir2 Family of NAD-dependent Protein Deacetylases

The regulation of protein function is often achieved through post-translational modifications including phosphorylation, methylation, ubiquitination, and acetylation. The role of acetylation has been most extensively studied in the context of histones, but it is becoming increasingly evident that this modification now includes other proteins. The Sir2 family of NAD-dependent deacetylases was initially recognized as mediating gene silencing through histone deacetylation, but several family members display non-nuclear sub-cellular localization and deacetylate non-histone protein substrates. Although many structural and enzymatic studies of Sir2 proteins have been reported, how substrate recognition is achieved by this family of enzymes is unknown. Here we use in vitro deacetylase assays and a variety of potential substrates to examine the substrate specificity of yeast homologue Hst2. We show that Hst2 is specific for acetyl-lysine within proteins; it does not deacetylate small polycations such as acetyl-spermine or acetylated amino ter-mini of proteins. Furthermore we have found that Hst2 displays conformational rather than sequence specificity, preferentially deacetylating acetyl-lysine within unstructured regions of proteins. Our results suggest that this conformational requirement may be a general feature for substrate recognition in the Sir2 family.

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LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

Cell Death and Disease ◽

10.1038/s41419-021-03728-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

You-hong Wang ◽

Zhen Guo ◽

Liang An ◽

Yong Zhou ◽

Heng Xu ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Cancer ◽

Noncoding Rnas ◽

Dependent Protein Kinase ◽

Damage Repair ◽

Dependent Protein ◽

Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

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Heparan sulfate and heparin interactions with proteins

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0589 ◽

2015 ◽

Vol 12 (110) ◽

pp. 20150589 ◽

Cited By ~ 108

Author(s):

Maria C. Z. Meneghetti ◽

Ashley J. Hughes ◽

Timothy R. Rudd ◽

Helena B. Nader ◽

Andrew K. Powell ◽

...

Keyword(s):

Heparan Sulfate ◽

Ex Vivo ◽

Sequence Specificity ◽

Structure Activity ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Heparin Activity

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro , ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.

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Effects of modifications of α-crystallin on its chaperone and other properties

Biochemical Journal ◽

10.1042/bj20011512 ◽

2002 ◽

Vol 364 (3) ◽

pp. 711-717 ◽

Cited By ~ 38

Author(s):

Barry K. DERHAM ◽

John J. HARDING

Keyword(s):

Congenital Cataract ◽

Point Mutations ◽

Small Heat Shock Protein ◽

High Molecular Mass ◽

Lens Crystallins ◽

Post Translational Modifications ◽

Chaperone Function ◽

Arginine Residues

The role of α-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. α-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by ‘trapping’ the protein in a high-molecular-mass complex. However, during aging, the chaperone function of α-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts. Within the central part of the lens there is no turnover of damaged protein, and therefore post-translational modifications of α-crystallin accumulate that can reduce chaperone function; this is compounded in cataract lenses. Extensive in vitro glycation, carbamylation and oxidation all decrease chaperone ability. In the present study, we report the effect of the modifiers malondialdehyde, acetaldehyde and methylglyoxal, all of which are pertinent to cataract. Also modification by aspirin, which is known to delay cataract and other diseases, has been investigated. Recently, two point mutations of arginine residues were shown to cause congenital cataract. 1,2-Cyclohexanedione modifies arginine residues, and the extent of modification needed for a change in chaperone function was investigated. Only methylglyoxal and extensive modification by 1,2-cyclohexanedione caused a decrease in chaperone function. This highlights the robust nature of α-crystallin.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012517 ◽

2020 ◽

Vol 295 (23) ◽

pp. 7905-7922 ◽

Cited By ~ 4

Author(s):

Nadine Ait-Bouziad ◽

Anass Chiki ◽

Galina Limorenko ◽

Shifeng Xiao ◽

David Eliezer ◽

...

Keyword(s):

Lipid Binding ◽

Binding Properties ◽

Post Translational Modifications ◽

Unique Role ◽

Tyrosine Residues ◽

Normal Functions ◽

Tau Aggregation ◽

Β Sheet

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in β-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.

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PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Thrombosis and Haemostasis ◽

10.1160/th13-06-0484 ◽

2014 ◽

Vol 111 (03) ◽

pp. 508-517 ◽

Cited By ~ 30

Author(s):

Carol Dangelmaier ◽

Bhanu Kanth Manne ◽

Elizabetta Liverani ◽

Jianguo Jin ◽

Paul Bray ◽

...

Keyword(s):

Protein Kinase ◽

Protein A ◽

Human Platelets ◽

Clot Retraction ◽

Functional Responses ◽

Atp Secretion ◽

Dependent Protein ◽

Induced Aggregation

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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USP52 regulates DNA end resection and chemosensitivity through removing inhibitory ubiquitination from CtIP

Nature Communications ◽

10.1038/s41467-020-19202-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ming Gao ◽

Guijie Guo ◽

Jinzhou Huang ◽

Jake A. Kloeber ◽

Fei Zhao ◽

...

Keyword(s):

Parp Inhibition ◽

Dependent Manner ◽

Interacting Protein ◽

Post Translational Modifications ◽

Regulatory Complexity ◽

Dna End Resection ◽

End Resection

Abstract Human C-terminal binding protein (CtBP)–interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.

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NAD+-Dependent Deacetylase Hst1p Controls Biosynthesis and Cellular NAD+ Levels in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.7044-7054.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 7044-7054 ◽

Cited By ~ 97

Author(s):

Antonio Bedalov ◽

Maki Hirao ◽

Jeffrey Posakony ◽

Melisa Nelson ◽

Julian A. Simon

Keyword(s):

De Novo ◽

Critical Role ◽

Salvage Pathway ◽

De Novo Synthesis ◽

Array Analysis ◽

Binding Partner ◽

Dependent Protein ◽

New Protein

ABSTRACT Nicotine adenine dinucleotide (NAD+) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD+-dependent protein deacetylases. In the latter two processes, NAD+ is consumed and converted to ADP-ribose and nicotinamide. NAD+ levels can be maintained by regeneration of NAD+ from nicotinamide via a salvage pathway or by de novo synthesis of NAD+ from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD+-dependent deacetylase Hst1p as a sensor of NAD+ levels and regulator of NAD+ biosynthesis. Using transcript arrays, we show that low NAD+ states specifically induce the de novo NAD+ biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD+-dependent deacetylase activity of Hst1p represses de novo NAD+ biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD+ biosynthesis genes. The removal of HST1-mediated repression of the NAD+ de novo biosynthesis pathway leads to increased cellular NAD+ levels. Transcript array analysis shows that reduction in cellular NAD+ levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD+-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD+ in comparison to other NAD+-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD+ sensor that monitors and regulates cellular NAD+ levels.

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Fructose 2,6-bisphosphate biosynthesis and regulation of carbohydrate metabolism in Aspergillus oryzae

Canadian Journal of Microbiology ◽

10.1139/w97-129 ◽

1998 ◽

Vol 44 (1) ◽

pp. 6-11

Author(s):

Gandhi Rádis-Baptista ◽

David N. Urquizo Valdivia ◽

José Abrahão-Neto

Keyword(s):

Cyclic Amp ◽

Carbohydrate Metabolism ◽

Aspergillus Oryzae ◽

Culture Conditions ◽

Dependent Protein Kinase ◽

Fructose 1,6 Bisphosphatase ◽

Dependent Protein ◽

Regulation Of Carbohydrate Metabolism

The biosynthesis and role of fructose 2,6-bisphosphate (Fru-2,6-P2) in carbohydrate metabolism during induction of an amylolytic system in Aspergillus oryzae was studied. Fluctuations in Fru-2,6-P2 were not dependent on the external glucose concentration during induction, whereas the level of Fru-2,6-P2 increased significantly when the oxygen concentration was diminished. Phosphofructokinase II (PFK II) of A. oryzae was sensitive to phosphorylation in vitro by the catalytic subunit of cyclic AMP dependent protein kinase, which increased the Vmax (twofold), although the Km (0.7 mM) remained unchanged. Phosphofructokinase I was neither activated by micromolar Fru-2,6-P2 nor inhibited by high ATP concentrations. The activity of fructose-1,6-bisphosphatase (FBPase) was subject to strong inhibition by Fru-2,6-P2. Addition of glucose to cultures under gluconeogenic conditions caused a decrease of approximately 40% in the FBPase activity within 4 min. These results indicate that the effect of Fru-2,6-P2 in A. oryzae could preferentially control gluconeogenesis. The addition of 0.1 M glucose under gluconeogenic culture conditions also showed that Fru-2,6-P2 fluctuations appeared to be, at least in short term, more closely related to temporal changes in the hexose-6-phosphate concentration.Key words: Aspergillus oryzae; fructose-2,6-bisphosphate; phosphofructokinase II (PFK II); cyclic AMP; gluconeogenesis control.

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