A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

Salivary Adiponectin and Albumin Levels on the Gingival Conditions of Patients Undergoing Bariatric Surgery: A Cohort Study.

This study analyzed the salivary proteomics, adiponectin and albumin, related to weight loss and periodontitis in patients undergoing bariatric surgery. This study included fourteen patients with morbid obesity (body mass index, BMI > 40 kg/m2) who underwent bariatric surgery Roux-en-Y gastric bypass (RYGB) in System Health Public in Brazil. Data on demographic and anthropometric measures were extracted from medical records preoperatively and 6 and 12 months post-surgery. The variables assessed were: probing pocket depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP), and stimulated whole-mouth saliva. In this study, saliva samples were analyzed by electrophoresis and immunoblotting. The ELISA kit was used to measure the MMP8 levels to determine potential markers for obesity. Adiponectin and albumin levels were also evaluated. Weight loss was associated with significant changes in patients' periodontal clinical data. Although 7 out of 10 periodontal patients showed an increase in salivary adiponectin levels after root planning treatment, when analyzed by Western blotting, the increase was not statistically significant (21.1 ± 4.8 to 26.3 ± 9.4 arbitrary units, p > 0.99). There was no correlation between albumin levels and salivary adiponectin pre-surgery, nor 6 months or 12 months after surgery. Weight loss was not improved by low-grade inflammation in bariatric patients, since albumin levels were similar between periods. Periodontitis is an inflammatory disease that is modulated by several factors, among which adiponectin plays an important role for the treatment of periodontal disease.

HOXA9 forms a repressive complex with nuclear matrix-associated protein SAFB to maintain acute myeloid leukemia.

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)-chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ-associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.

Native chemical ligation approach to sensitively probe tissue acyl-CoA pools.

During metabolism, carboxylic acids are often activated by conjugation to the thiol of coenzyme A (CoA). The resulting acyl-CoAs comprise a group of ∼100 thioester-containing metabolites that could modify protein behavior through non-enzymatic N-acylation of lysine residues. However, the importance of many potential acyl modifications remains unclear because antibody-based methods to detect them are unavailable and the in vivo concentrations of their respective acyl-CoAs are poorly characterized. Here, we develop cysteine-triphenylphosphonium (CysTPP), a mass spectrometry probe that uses "native chemical ligation" to sensitively detect the major acyl-CoAs present in vivo through irreversible modification of its amine via a thioester intermediate. Using CysTPP, we show that longer-chain (C13-C22) acyl-CoAs often constitute ∼60% of the acyl-CoA pool in rat tissues. These hydrophobic longer-chain fatty acyl-CoAs have the potential to non-enzymatically modify protein residues.

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.

Nucleotide-binding sites can enhance N-acylation of nearby protein lysine residues.

Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. N-acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S-acylation, as proximity leads to rapid S → N-transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N-transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N-acetylation in vitro. Furthermore, by generating modelled structures of proteins N-acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N-acetylation and identify where nucleotide binding could enhance N-acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N-malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N-acetylation and N-succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N-malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N-acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N-acylation in vivo.

Agonists of Orally Expressed TRP Channels Stimulate Salivary Secretion and Modify the Salivary Proteome.

Natural compounds that can stimulate salivary secretion are of interest in developing treatments for xerostomia, the perception of a dry mouth, that affects between 10 and 30% of the adult and elderly population. Chemesthetic transient receptor potential (TRP) channels are expressed in the surface of the oral mucosa. The TRPV1 agonists capsaicin and piperine have been shown to increase salivary flow when introduced into the oral cavity but the sialogogic properties of other TRP channel agonists have not been investigated. In this study we have determined the influence of different TRP channel agonists on the flow and protein composition of saliva. Mouth rinsing with the TRPV1 agonist nonivamide or menthol, a TRPM8 agonist, increased whole mouth saliva (WMS) flow and total protein secretion compared with unstimulated saliva, the vehicle control mouth rinse or cinnamaldehyde, a TRPA1 agonist. Nonivamide also increased the flow of labial minor gland saliva but parotid saliva flow rate was not increased. The influence of TRP channel agonists on the composition and function of the salivary proteome was investigated using a multi-batch quantitative MS method novel to salivary proteomics. Inter-personal and inter-mouth rinse variation was observed in the secreted proteomes and, using a novel bioinformatics method, inter-day variation was identified with some of the mouth rinses. Significant changes in specific salivary proteins were identified after all mouth rinses. In the case of nonivamide, these changes were attributed to functional shifts in the WMS secreted, primarily the over representation of salivary and nonsalivary cystatins which was confirmed by immunoassay. This study provides new evidence of the impact of TRP channel agonists on the salivary proteome and the stimulation of salivary secretion by a TRPM8 channel agonist, which suggests that TRP channel agonists are potential candidates for developing treatments for sufferers of xerostomia.

Wine astringency reduces flavor intensity of Brussels sprouts.

The bitterness of vegetables is a leading reason why they are avoided by children and some adults. Bitterness is perceived via TAS2R receptors located on the tongue. In contrast, astringency is a mouthfeel rather than a taste, and is perceived as a dry, puckering sensation. To date few reports have suggested any interactions between the two processes even though they often occur simultaneously in many real foods. In this study, we have used Brussels sprouts as an exemplar bitter vegetable and examined the influence of a number of different interventions on perceived intensity. Subjects rated the intensity of Brussels sprouts before and after three interventions: gravy, red wine, and water. Only red wine caused a significant (p < .0001) decrease in VAS scale, from 5.5 to 3.5 on a 10-point labeled magnitude scale. The results suggest the astringency of the red wine affected the perception of bitter in the Brussels sprout. Some possible mechanisms are discussed. PRACTICAL APPLICATIONS: This report reveals a possible insight into how bitterness is perceived in humans. By using astringency to affect salivary proteins, we suggest they may play a role in the detection of bitterness. This may be by helping to transport bitterness compounds to the taste bud receptors or a separate mechanism. Potentially this also opens up new ways to block bitterness.