HDAC8 Catalyzes the Hydrolysis of Long Chain Fatty Acyl Lysine

The histone deacetylase (HDAC) family regulates many biological pathways through the deacetylation of lysine residues on histone and nonhistone proteins. Mammals have 18 HDACs that are classified into four classes. Class I, II, and IV are zinc-dependent, while class III is nicotinamide adenine dinucleotide (NAD⁺)-dependent lysine deacetylase or sirtuins. HDAC8, a class I HDAC family member, has been shown to have low deacetylation activity compared to other HDACs <i>in vitro.</i> Recent studies showed that several sirtuins, with low deacetylase activities, can actually hydrolyze other acyl lysine modifications more efficiently. Inspired by this, we tested the activity of HDAC8 using a variety of different acyl lysine peptides. Screening a panel of peptides with different acyl lysine modifications, we found that HDAC8 can catalyze the removal of acyl groups with 2–16 carbons from lysine 9 of the histone H3 peptide (H3K9). Interestingly, the catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of HDAC8 on octanoyl, dodecanoyl, and myristoyl lysine are several-fold better than that on acetyl lysine. The increased catalytic efficiencies of HDAC8 on larger fatty acyl groups are due to the much lower <i>K</i>_m values. T-cell leukemia Jurkat cells treated with a HDAC8 specific inhibitor, PCI-34051, exhibited an increase in global fatty acylation compared to control treatment. Thus, the de-fatty-acylation activity of HDAC8 is likely physiologically relevant. This is the first report of a zinc-dependent HDAC with de-fatty-acylation activity, and identification of HDAC8 de-fatty-acylation targets will help to further understand the function of HDAC8 and protein lysine fatty acylation

MS/MS of Synthetic Peptide Is Not Sufficient to Confirm New Types of Protein Modifications

Protein post-translational modification (PTM) is one of the major regulatory mechanisms that fine-tune protein functions. Undescribed mass shifts, which may suggest novel types of PTMs, continue to be discovered because of the availabilities of more sensitive mass spectrometry technologies and more powerful sequence alignment algorithms. In this study, the histone extracted from HeLa cells was analyzed using an approach that takes advantages of in vitro propionylation, efficient peptide separation using isoelectric focusing fractionation, and the high sensitivity of the linear ion trap coupled with hybrid FT mass spectrometer. One modified peptide was identified with a new type of protein modification (+42 Da), which was assigned to acetylation of threonine 15 in histone2A. The modified peptide was verified by careful manual evaluation of the tandem mass spectrum and confirmed by high-resolution MS/MS analysis of the corresponding synthetic peptide. However, HPLC coelution and MS/MS/MS of key ions showed that the +42 Da mass shifts at threonine residue did not correspond to acetylation. The key fragment ion, y4, in the MS/MS/MS spectra (indicative of the modification site) differed between the in vivo and synthetic peptide. We showed that the misidentification was originated from sequence homologues and chemical derivitization during sample preparation. This result indicated that a more stringent procedure that includes MS/MS, MS/MS/MS, and HPLC coelution of synthetic peptides is required to identify a new PTM

SAHA Regulates Histone Acetylation, Butyrylation, and Protein Expression in Neuroblastoma

Emerging evidence suggests that suberoylanilide hydroxamic acid (SAHA), a clinically approved HDAC inhibitor for cutaneous T-cell lymphoma, shows promising clinical benefits in neuroblastoma, the most common extra cranial solid neoplasm with limited choice of therapeutic intervention. However, the molecular mechanism under which the compound exerts its antitumor effect remains elusive. Here we report a quantitative proteomics study that determines changes of protein expression, histone lysine acetylation, and butyrylation in response to SAHA treatment. We detected and quantified 28 histone lysine acetylation and 18 histone lysine butyrylation marks, most of which are dramatically induced by SAHA. Importantly, we identified 11 histone K_bu sites as novel histone marks in human cells. Furthermore, quantitative proteomic analysis identified 5426 proteins, among which 510 proteins were up-regulated and 508 proteins were down-regulated (significant <i>p</i> value <0.05). The subsequent bioinformatics analysis identified distinct SAHA-response gene ontology (GO) categories and signaling pathways, including cellular metabolism and DNA-dependent pathways. Our study therefore reveals new histone epigenetic marks and offers key insights into the molecular mechanism by which SAHA regulates proteomic changes in neuroblastoma cells and identifies biomarker candidates for SAHA

The accuracy and safety of injection.

<p>(A), (B) Representative images of methylene-blue injection. The yellow arrows pointed to injection sites, all of the sites were around the desired myocardial ischemic region (pale region). The red arrow pointed to the ligation site of the LAD. The myocardial ischemic region was marked with red curve. No MB staining was detected in the pericardium. (C) Representative image of the echocardiography. During the mapping and injection, the pericardial effusion was not detected in two groups. (D) Representative image of the surface electrocardiogram and intracardiac electrical recording. The operation did not cause malignant arrhythmia in real time.</p

HGF expression.

<p>(A) Representative image of HGF expression. (B) Bar graph showed HGF protein had higher expression in the infarct and peri-infarct zone of the treatment group compared to that in saline group (*P<0.01). (C) In HGF group, the myocardial infarct and peri-infarct zone had higher HGF expression than the normal zone (*P<0.01, ^#P<0.01). I, infarct zone; P, peri-infarct zone; N, normal zone.</p

Injection of speed and depth.

<p>Yes: the overflow and bubbling were detected in the endocardium. No: the overflow and bubbling were not detected in the endocardium, or MB staining not in the pericardium.</p

Infarct size and myocardial voltage.

<p>(A), (B) the left ventricular geometry before and after the GTx. Gray and purple respectively indicates the myocardial infarct and normal zones; the region between gray and purple is transitional zone. Red arrow indicates injected site marked with white dots. (C) Bar graph showed the infarct sizes significantly reduced in the HGF-injected hearts after GTx than those before GTx. (D), (E) Bar graph explained the myocardial voltage levels in the infarct and normal zones of HGF-injected hearts. There were significantly higher myocardial voltage levels in the infarct zone of HGF-injected hearts after GTx than those before GTx. *P<0.01 vs. before GTx in HGF group.</p

The analyses of clinical chemistry.

<p>G: groups; S: saline; H: HGF; T: time; B: before the LAD ligation; L1 h, L6 H, L24 h, L3 d: 1, 6, 24 hours and3 days after the LAD ligation; T0 h: before the injection; T24 h, T3 d, T4 w: 24 hours, 3 days and 4 weeks after the injection. *P<0.01 vs. before the ligation.</p

The intramyocardial injection system.

<p>(A)The injection device comprises a catheter and handle. The catheter involves in mapping and injection functions. (B) The distal end of the catheter is equipped with tip and ring electrodes, a location sensor and a 27 guage needle designed for transendocardial injection. The red circle marks out a drop of saline injected through the needle tip. (C) The locker fixed the needle whose length was selected. (D) The adjusting knob controls the length of needle into myocardium. Two Luer fit for connection to an auto-syringe pump and a syringe of contrast medium, respectively.</p

The vascular density.

<p>(A) Representative pictures of Lectin stained sections. (B) The immunofluorescence of the Ki67-positive cells in the border zone of the infarct. (C) Bar graph showing the quantification of microvessels. (D) Bar graph showing the quantification of Ki67-positive vessels. *P<0.01 vs. saline group.</p