Enzyme Structure and Mechanism (Second Edition)

Herries, DG

doi:10.1016/0307-4412(85)90213-4

Cited by 17 publications

(5 citation statements)

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“…To assess if measured activities on isolated peptides appropriately reflects HDAC6 activity on the natural substrate protein, we measured the activity of HDAC6 towards a full-length, singly-acetylated histone protein and a respective 13-mer peptide analog; catalytic efficiency was similar, albeit higher for the peptide: 14 × 10 4 M −1 s −1 vs. 8 × 10 4 M −1 s −1 for the full-length substrate, mostly due to an increased value of k cat (Table S2 ). Based on these results and the observation that enzymes that are catalytically efficient in the cell have a k cat / K M value of at least 10 4 M −1 s −1 28 , we defined this value as a cutoff for distinguishing substrate and slow substrate or non-substrate peptides. In cells, HDAC6 selectivity will be determined by the relative reactivity, so substrates with high values of k cat / K M will out-compete those with low values if present at similar concentrations.…”

Section: Resultsmentioning

confidence: 99%

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Varga

Diffley

Leng

et al. 2022

Sci Rep

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Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

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Section: Resultsmentioning

confidence: 99%

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Varga

Diffley

Leng

et al. 2022

Sci Rep

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“…*P<0.0002 (one tailed, unpaired t test), calculated using the values at 160 nM IFIH1. Bound fraction was calculated as in ref 12 , and fitted with the Hill equation 29 . The dissociation constants ( K d ) obtained from curve fitting are shown on the right.…”

Section: Figmentioning

confidence: 99%

Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling

et al. 2014

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The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, and of other patients with undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (MDA5) cause a spectrum of neuro-immunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer a gain-of-function - so that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

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“…The experiments were performed using various guanine nucleotide substrate concentrations in the presence of constant amounts of either 100 M pppGpp or ppGpp. The k cat , K m , and specificity constant k cat /K m were calculated to determine substrate preferences of RelQ Ef (42). RelQ Ef has a moderate preference toward GDP as a substrate over GTP, but the strength of this preference (k cat /K m ) is dependent on the nature of the alarmone present.…”

Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 99%

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

et al. 2015

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The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel Ef ) and the small alarmone synthetase (SAS) RelQ Ef . Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ Ef synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQ Ef also efficiently utilized GMP to form GMP 3=-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ Ef was activated only by ppGpp. Furthermore, enzymatic activity of RelQ Ef is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQ Ef ), we found that, in addition to (p)ppGpp, RelQ Ef is an efficient producer of pGpp (GMP 3=-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQ Ef and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones. In order to survive under adverse environmental conditions, such as nutrient starvation, bacteria have evolved complex, interconnected regulatory networks that sense and integrate internal and external metabolic cues to activate cellular responses that enhance bacterial survival. One critical and highly conserved mechanism employed by bacteria to cope with nutritional as well as a variety of environmental and chemical stresses is the stringent response (SR). The SR is mediated by the accumulation of two guanine analog...

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Enzyme Structure and Mechanism (Second Edition)

Cited by 17 publications

References 0 publications

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

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