Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A3 Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7.

This study describes the localization and computational prediction of a binding site for the A3 adenosine receptor (A3AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y2847.55 in TMD7 and Y2938.54 in H8 and by π-NH bonding between Y2847.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G291.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y2938.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A3AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A3AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A3AR positive allosteric modulators.

Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

Ure(k)a! Sirtuins Regulate Mitochondria.

Increasing evidence suggests that multiple metabolic pathways are regulated by sirtuin-dependent protein deacetylation in the mitochondria. In this issue, Nakagawa et al. (2009) show that the sirtuin SIRT5 deacetylates and activates a mitochondrial enzyme, carbamoyl phosphate synthetase 1, which mediates the first step in the urea cycle.

Current Trends in Sirtuin Activator and Inhibitor Development.

Sirtuins are NAD+-dependent protein deacylases and key metabolic regulators, coupling the cellular energy state with selective lysine deacylation to regulate many downstream cellular processes. Humans encode seven sirtuin isoforms (Sirt1-7) with diverse subcellular localization and deacylase targets. Sirtuins are considered protective anti-aging proteins since increased sirtuin activity is canonically associated with lifespan extension and decreased activity with developing aging-related diseases. However, sirtuins can also assume detrimental cellular roles where increased activity contributes to pathophysiology. Modulation of sirtuin activity by activators and inhibitors thus holds substantial potential for defining the cellular roles of sirtuins in health and disease and developing therapeutics. Instead of being comprehensive, this review discusses the well-characterized sirtuin activators and inhibitors available to date, particularly those with demonstrated selectivity, potency, and cellular activity. This review also provides recommendations regarding the best-in-class sirtuin activators and inhibitors for practical research as sirtuin modulator discovery and refinement evolve.

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis.

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.

Chemical Regulation of the Protein Quality Control E3 Ubiquitin Ligase C-Terminus of Hsc70 Interacting Protein (CHIP).

The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) is an important regulator of proteostasis. Despite playing an important role in maintaining proteostasis, little progress has been made in developing small molecules that regulate ubiquitin transfer by CHIP. Here we used differential scanning fluorimetry to identify compounds that bound CHIP. Compounds that bound CHIP were then analyzed by quantitative ubiquitination assays to identify those that altered CHIP function. One compound, MS.001, inhibited both the chaperone binding and ubiquitin ligase activity of CHIP at low micromolar concentrations. Interestingly, we found that MS.001 did not have activity against isolated U-box or tetratricopeptide (TPR) domains, but instead only inhibited full-length CHIP. Using in silico docking we identified a potential MS.001 binding site on the linker domain of CHIP and mutation of this site rendered CHIP resistant to MS.001. Together our data identify an inhibitor of the E3 ligase CHIP and provides insight into the development of compounds that regulate CHIP activity.

Human sirtuins are differentially sensitive to inhibition by nitrosating agents and other cysteine oxidants.

Sirtuins (e.g. human Sirt1-7) catalyze the removal of acyl groups from lysine residues in proteins in an NAD+-dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Although multiple reports suggest that sirtuin activity is regulated by oxidative post-translational modifications of cysteines during inflammation and aging, no systematic comparative study of potential direct sirtuin cysteine oxidative modifications has been performed. Here, using IC50 and kinact/KI analyses, we quantified the ability of nitrosothiols (S-nitrosoglutathione and S-nitroso-N-acetyl-d,l-penicillamine), nitric oxide, oxidized GSH, and hydrogen peroxide to post-translationally modify and inhibit the deacylase activity of Sirt1, Sirt2, Sirt3, Sirt5, and Sirt6. The inhibition was correlated with cysteine modification and assessed with chemical-probe and blot-based assays for cysteine S-nitrosation, sulfenylation, and glutathionylation. We show that the primarily nuclear sirtuins Sirt1 and Sirt6, as well as the primarily cytosolic sirtuin Sirt2, are modified and inhibited by cysteine S-nitrosation in response to exposure to both free nitric oxide and nitrosothiols (kinact/KI ≥ 5 m-1 s-1), which is the first report of Sirt2 and Sirt6 inhibition by S-nitrosation. Surprisingly, the mitochondrial sirtuins Sirt3 and Sirt5 were resistant to inhibition by cysteine oxidants. Collectively, these results suggest that nitric oxide-derived oxidants may causatively link nuclear and cytosolic sirtuin inhibition to aging-related inflammatory disease development.

Mitochondrial Metabolic Reprogramming by CD36 Signaling Drives Macrophage Inflammatory Responses.

RATIONALE: A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. OBJECTIVE: To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways. METHODS AND RESULTS: Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-κB (nuclear factor-κB) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-κB activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages. CONCLUSIONS: These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

ICEKAT: an interactive online tool for calculating initial rates from continuous enzyme kinetic traces.

BACKGROUND: Continuous enzyme kinetic assays are often used in high-throughput applications, as they allow rapid acquisition of large amounts of kinetic data and increased confidence compared to discontinuous assays. However, data analysis is often rate-limiting in high-throughput enzyme assays, as manual inspection and selection of a linear range from individual kinetic traces is cumbersome and prone to user error and bias. Currently available software programs are specialized and designed for the analysis of complex enzymatic models. Despite the widespread use of initial rate determination for processing kinetic data sets, no simple and automated program existed for rapid analysis of initial rates from continuous enzyme kinetic traces. RESULTS: An Interactive Continuous Enzyme Kinetics Analysis Tool (ICEKAT) was developed for semi-automated calculation of initial rates from continuous enzyme kinetic traces with particular application to the evaluation of Michaelis-Menten and EC50/IC50 kinetic parameters, as well as the results of high-throughput screening assays. ICEKAT allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining ranges to fit in general purpose programs like Microsoft Excel and Graphpad Prism, while still maintaining simplicity in determining initial rates. As a test case, we quickly analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of the protein lysine deacetylase SIRT1 to small-molecule activators. CONCLUSIONS: ICEKAT allows simultaneous visualization of individual initial rate fits and the resulting Michaelis-Menten or EC50/IC50 kinetic model fits, as well as hits from high-throughput screening assays. In addition to serving as a convenient program for practicing enzymologists, ICEKAT is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated Michaelis-Menten or EC50/IC50 kinetic parameters. For the convenience of the research community, we have made ICEKAT freely available online at https://icekat.herokuapp.com/icekat.

Development of activity-based probes for the protein deacylase Sirt1.

Sirtuins are NAD+-dependent protein deacylases that remove acyl modifications from acyl-lysine residues, resulting in essential cellular signaling. Recognized for their role in lifespan extension, humans encode seven sirtuin isoforms (Sirt1-7), and loss of sirtuin deacylase activity is implicated in many aging-related diseases. Despite being intriguing therapeutic targets, cellular studies of sirtuins are hampered by the lack of chemical probes to measure sirtuin activity independent of sirtuin protein levels. Here, we use a modular, peptide-based approach to develop activity-based probes (ABPs) that directly measure Sirt1 activity in vitro and in cell lysates. ABPs were synthesized containing four elements: (1) thioacetyl-lysine for mechanism-based affinity towards only active sirtuins, (2) either histone H3 lysine-14 (H3K14) or p53 sequences for Sirt1 specificity, (3) a diazirine for covalent labeling upon UV irradiation, and (4) an alkyne for bioorthogonal conjugation to a fluorophore for gel-based detection of active Sirt1. Compared to the H3K14 ABP, the p53 ABP showed increased sensitivity and selective labeling of active Sirt1. Acyl-lysine peptide competition, pharmacological inhibition, and inhibitory post-translational modification of Sirt1 resulted in the loss of p53 ABP labeling both in vitro and in HEK293T cell lysates, consistent with the ABP measuring decreased Sirt1 activity. Furthermore, the p53 ABP measured subcellular Sirt1 activity in MCF7 breast cancer cells. The development of a Sirt1-selective ABP that detects Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches demonstrates the utility of a modular, peptide-based approach for selective-targeting of the sirtuin protein family and provides a framework for further development of sirtuin-selective chemical probes.

Nitric oxide antagonism to glioblastoma photodynamic therapy and mitigation thereof by BET bromodomain inhibitor JQ1.

Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells up-regulate iNOS after a photodynamic challenge and that the resulting NO not only increases resistance to apoptosis but renders surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/RelA by bromodomain and extra-terminal (BET) protein Brd4 is crucial; and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained. (i) Like iNOS, Brd4 protein and p65-acK levels increased severalfold in photostressed cells. (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK up-regulation after PDT but strongly suppressed iNOS, survivin, and Bcl-xL up-regulation, along with the growth and invasion spurt of PDT-surviving cells. (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition. (iv) Finally, at 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy.

Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes.

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (•NO)-derived agents. S-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical •NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.

Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction.

Emerging evidence suggests that protein acetylation is a broad-ranging regulatory mechanism. Here we utilize acetyl-peptide arrays and metabolomic analyses to identify substrates of mitochondrial deacetylase Sirt3. We identified ornithine transcarbamoylase (OTC) from the urea cycle, and enzymes involved in ß-oxidation. Metabolomic analyses of fasted mice lacking Sirt3 (sirt3(-/-)) revealed alterations in ß-oxidation and the urea cycle. Biochemical analysis demonstrated that Sirt3 directly deacetylates OTC and stimulates its activity. Mice under caloric restriction (CR) increased Sirt3 protein levels, leading to deacetylation and stimulation of OTC activity. In contrast, sirt3(-/-) mice failed to deacetylate OTC in response to CR. Inability to stimulate OTC under CR led to a failure to reduce orotic acid levels, a known outcome of OTC deficiency. Thus, Sirt3 directly regulates OTC activity and promotes the urea cycle during CR, and the results suggest that under low energy input, Sirt3 modulates mitochondria by promoting amino acid catabolism and ß-oxidation.

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy.

An academic chemical screening approach was developed by using 2D protein-detected NMR, and a 352-chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric-difference intensity analysis (DIA)-was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3-1.6 mm. DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.

Metabolically Derived Lysine Acylations and Neighboring Modifications Tune the Binding of the BET Bromodomains to Histone H4.

Recent proteomic studies discovered histone lysines are modified by acylations beyond acetylation. These acylations derive from acyl-CoA metabolites, potentially linking metabolism to transcription. Bromodomains bind lysine acylation on histones and other nuclear proteins to influence transcription. However, the extent bromodomains bind non-acetyl acylations is largely unknown. Also unclear are the effects of neighboring post-translational modifications, especially within heavily modified histone tails. Using peptide arrays, binding assays, sucrose gradients, and computational methods, we quantified 10 distinct acylations for binding to the bromodomain and extraterminal domain (BET) family. Four of these acylations (hydroxyisobutyrylation, malonylation, glutarylation, and homocitrullination) had never been tested for bromodomain binding. We found N-terminal BET bromodomains bound acetylated and propionylated peptides, consistent with previous studies. Interestingly, all other acylations inhibited binding of the BET bromodomains to peptides and nucleosomes. To understand how context tunes bromodomain binding, effects of neighboring methylation, phosphorylation, and acylation within histone H4 tails were determined. Serine 1 phosphorylation inhibited binding of the BRD4 N-terminal bromodomain to polyacetylated H4 tails by >5-fold, whereas methylation had no effect. Furthermore, binding of BRDT and BRD4 N-terminal bromodomains to H4K5acetyl was enhanced 1.4-9.5-fold by any neighboring acylation of lysine 8, indicating a secondary H4K8acyl binding site that is more permissive of non-acetyl acylations than previously appreciated. In contrast, C-terminal BET bromodomains exhibited 9.9-13.5-fold weaker binding for polyacylated than for monoacylated H4 tails, indicating the C-terminal bromodomains do not cooperatively bind multiple acylations. These results suggest acyl-CoA levels tune or block recruitment of the BET bromodomains to histones, linking metabolism to bromodomain-mediated transcription.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment.

Nitrosothiol formation and S-nitrosation signaling through nitric oxide synthases.

Nitric oxide (NO) is a gaseous signaling molecule impacting many biological pathways. NO is produced in mammals by three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS and eNOS produce low concentrations of NO for paracrine signaling; NO produced and released from one cell diffuses to a neighboring cell where it binds and activates soluble guanylyl cyclase (sGC). iNOS produces high concentrations of NO using NO toxicity to amplify the innate immune response. Recent work has also defined protein cysteine S-nitrosation as a pathway of sGC-independent NO signaling. Though many studies have shown that S-nitrosation regulates the activity of NOS isoforms and other proteins in vivo, many issues need to be resolved to establish S-nitrosation as a viable signaling mechanism. Several chemical mechanisms result in S-nitrosation including transition metal-catalyzed pathways, NO oxidation followed by thiolate reaction, and thiyl radical recombination with NO. Once formed, nitrosothiols can be transferred between cellular cysteine residues via transnitrosation reactions. However, it is largely unclear how these chemical processes result in selective S-nitrosation of specific cellular cysteine residues. S-nitrosation site selectivity may be imparted via direct interactions or colocalization with NOS isoforms that focus chemical or transnitrosation mechanisms of nitrosothiol formation or transfer. Here, we discuss chemical mechanisms of nitrosothiol formation, S-nitrosation of NOS isoforms, and potential S-nitrosation signaling cascades resulting from NOS S-nitrosation.

A multi-level modeling approach examining PTSD symptom reduction during prolonged exposure therapy: moderating effects of number of trauma types experienced, having an HIV-related index trauma, and years since HIV diagnosis among HIV-positive adults.

People living with HIV (PLWH) have extensive interpersonal trauma histories and higher rates of posttraumatic stress disorder (PTSD) than the general population. Prolonged exposure (PE) therapy is efficacious in reducing PTSD across a variety of trauma samples; however, research has not examined factors that influence how PTSD symptoms change during PE for PLWH. Using multi-level modeling, we examined the potential moderating effect of number of previous trauma types experienced, whether the index trauma was HIV-related or not, and years since HIV diagnosis on PTSD symptom reduction during a 10-session PE protocol in a sample of 51 PLWH. In general, PTSD symptoms decreased linearly throughout the PE sessions. Experiencing more previous types of traumatic events was associated with a slower rate of PTSD symptom change. In addition, LOCF analyses found that participants with a non-HIV-related versus HIV-related index trauma had a slower rate of change for PTSD symptoms over the course of PE. However, analyses of raw data decreased this finding to marginal. Years since HIV diagnosis did not impact PTSD symptom change. These results provide a better understanding of how to tailor PE to individual clients and aid clinicians in approximating the rate of symptom alleviation. Specifically, these findings underscore the importance of accounting for trauma history and index trauma type when implementing a treatment plan for PTSD in PLWH.