BACKGROUND AND PURPOSE: N-methyl-d-aspartate receptor (NMDAR) and leucine-rich glioma-inactivated protein 1 (LGI1) encephalitis are important types of autoimmune encephalitis (AE) with significant morbidity. In this study, we used a proteomic approach in search of novel clinically relevant biomarkers in these types of encephalitides. METHODS: Swedish and Czech tertiary neuroimmunology centers collaborated in this retrospective exploratory study. Fifty-eight cerebrospinal fluid (CSF) samples of 28 patients with AE (14 definite NMDAR, 14 with definite LGI1 encephalitis) and 30 controls were included. CSF samples were analyzed using proximity extension assay technology (Olink Target 96 Inflammation panel). For each CSF sample, 92 proteins were measured. Clinical variables were retrospectively collected, and correlations with protein levels were statistically analyzed. RESULTS: Patients and controls differed significantly in the following 18 biomarkers: TNFRSF9, TNFRSF12, TNFRSF14, TNFβ, TNFα, IL7, IL10, IL12B, IFNγ, CD5, CD6, CASP8, MMP1, CXCL8, CXCL10, CXCL11, IL20RA, and sirtuin 2 (SIRT2). In LGI1 encephalitis, no clinically useful association was found between biomarkers and clinical variables. In the NMDAR encephalitis group, SIRT2, TNFβ, and CD5 were significantly associated with ovarian teratoma. For SIRT2, this was true even for the first patients' CSF sample (SIRT2 without vs. with tumor, mean ± SD = 2.2 ± 0.29 vs. 2.88 ± 0.48; p = 0.007, 95% confidence interval = -1.15 to -0.22; r statistic in point-biserial correlation (rpb) = 0.66, p = 0.011). SIRT2 was positively correlated with age (rpb = 0.39, p = 0.018) and total hospital days (r = 0.55, p = <0.001). CONCLUSIONS: SIRT2 should be investigated as a biomarker of paraneoplastic etiology in NMDAR encephalitis.
- Keywords
- anti-N-methyl-d-aspartate receptor encephalitis, encephalitis, limbic encephalitis, paraneoplastic syndromes, sirtuin 2,
- MeSH
- Autoantibodies MeSH
- Biomarkers cerebrospinal fluid MeSH
- Anti-N-Methyl-D-Aspartate Receptor Encephalitis * cerebrospinal fluid MeSH
- Encephalitis MeSH
- Hashimoto Disease MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Proteomics MeSH
- Retrospective Studies MeSH
- Sirtuin 2 MeSH
- Check Tag
- Humans MeSH
- Infant, Newborn MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Autoantibodies MeSH
- Biomarkers MeSH
- SIRT2 protein, human MeSH Browser
- Sirtuin 2 MeSH
Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.
- Keywords
- bovine serum albumin effect, continuous activity assay, fluorescence quenching, histone deacetylases, myristoylated substrates, sirtuin inhibitors, sirtuins,
- MeSH
- Coloring Agents MeSH
- Lysine MeSH
- Peptides MeSH
- Sirtuin 1 metabolism MeSH
- Sirtuin 2 metabolism MeSH
- Sirtuin 3 * metabolism MeSH
- Sirtuins * metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Coloring Agents MeSH
- Lysine MeSH
- Peptides MeSH
- Sirtuin 1 MeSH
- Sirtuin 2 MeSH
- Sirtuin 3 * MeSH
- Sirtuins * MeSH
Zinc-dependent histone deacetylases (HDACs) and sirtuins (SIRT) represent two different classes of enzymes which are responsible for deacylation of modified lysine side chains. The repertoire of acyl residues on lysine side chains identified in vivo is rapidly growing, and very recently lysine lactoylation was described to be involved in metabolic reprogramming. Additionally, lysine pyruvoylation represents a marker for aging and liver cirrhosis. Here, we report a systematic analysis of acyl-specificity of human zinc-dependent HDAC and sirtuin isoforms. We identified HDAC3 as a robust delactoylase with several-thousand-fold higher activity as compared to SIRT2, which was claimed to be the major in vivo delactoylase. Additionally, we systematically searched for enzymes, capable of removing pyruvoyl residues from lysine side chains. Using model peptides, we uncovered high depyruvoylase activity for HDAC6 and HDAC8. Interestingly, such substrates have extremely low KM values for both HDAC isoforms, pointing to possible in vivo functions.
- MeSH
- Histone Deacetylases MeSH
- Histone Deacetylase Inhibitors MeSH
- Humans MeSH
- Lysine * chemistry MeSH
- Protein Isoforms MeSH
- Repressor Proteins metabolism MeSH
- Sirtuin 2 * MeSH
- Aging MeSH
- Zinc MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Systematic Review MeSH
- Names of Substances
- HDAC8 protein, human MeSH Browser
- Histone Deacetylases MeSH
- Histone Deacetylase Inhibitors MeSH
- Lysine * MeSH
- Protein Isoforms MeSH
- Repressor Proteins MeSH
- Sirtuin 2 * MeSH
- Zinc MeSH
