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 peptidome analysis and protease prediction during orthodontic treatment with fixed appliances.

Orthodontic tooth movement (OTM) occurs through proteolytic remodelling within the periodontium following the application of external force to the tooth. This study describes the first characterization of the salivary peptidome and protease profile during the alignment stage of fixed appliance orthodontic treatment. Unstimulated whole mouth saliva from 16 orthodontic patients (10 males, 6 females, mean (SD) age 15.2 (1.6) years) was collected prior to fixed appliance placement (T1), 1-h (T2), 1-week (T3) following fixed appliance placement and on completion of mandibular arch alignment (T4). Salivary peptides were extracted using filtration followed by mass spectrometry to identify amino acid sequences. Protease prediction was carried out in silico using Proteasix and validated with gelatin zymography and enzyme-linked immunosorbent assay. A total of 2852 naturally-occurring peptides were detected, originating from 436 different proteins. Both collagen and statherin-derived peptide levels were increased at T2. Proteasix predicted 73 proteases potentially involved in generating these peptides, including metalloproteinases, calpains and cathepsins. Changes in predicted activity of proteases over time were also observed, with most metalloproteinases showing increased predicted activity at T2-T3. Increased gelatinolytic activity and MMP8/MMP9 levels were detected at T3. Collectively, multiple protein targets and changes in protease-predicted activity during OTM have been identified.

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.

Comparative Cell Surface Proteomic Analysis of the Primary Human T Cell and Monocyte Responses to Type I Interferon.

The cellular response to interferon (IFN) is essential for antiviral immunity, IFN-based therapy and IFN-related disease. The plasma membrane (PM) provides a critical interface between the cell and its environment, and is the initial portal of entry for viruses. Nonetheless, the effect of IFN on PM proteins is surprisingly poorly understood, and has not been systematically investigated in primary immune cells. Here, we use multiplexed proteomics to quantify IFNα2a-stimulated PM protein changes in primary human CD14+ monocytes and CD4+ T cells from five donors, quantifying 606 and 482 PM proteins respectively. Comparison of cell surface proteomes revealed a remarkable invariance between donors in the overall composition of the cell surface from each cell type, but a marked donor-to-donor variability in the effects of IFNα2a. Furthermore, whereas only 2.7% of quantified proteins were consistently upregulated by IFNα2a at the surface of CD4+ T cells, 6.8% of proteins were consistently upregulated in primary monocytes, suggesting that the magnitude of the IFNα2a response varies according to cell type. Among these differentially regulated proteins, we found the viral target Endothelin-converting enzyme 1 (ECE1) to be an IFNα2a-stimulated protein exclusively upregulated at the surface of CD4+ T cells. We therefore provide a comprehensive map of the cell surface of IFNα2a-stimulated primary human immune cells, including previously uncharacterized interferon stimulated genes (ISGs) and candidate antiviral factors.

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.

ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex.

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.

Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex.

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)-1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III-like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.

Most people suffer from occasional cold sores, which are caused by the herpes simplex virus. This virus causes infections that last your entire life, but for the most part it lies dormant in your cells and reactivates only at times of stress. When it reactivates, the virus manipulates host cells to make new virus particles that may spread the infection to other people. Like many other viruses, herpes simplex viruses also steal jelly-like structures known as membranes from their host cells to form protective coats around new virus particles. In cells from humans and other animals, proteins belonging to a molecular machine known as ESCRT form filaments that bend and break membranes as the cells require. Many viruses hijack the ESCRT machinery to wrap membranes around new virus particles. However, herpes simplex viruses do not follow the usual rules for activating this machine. Instead, they rely on two viral proteins called pUL7 and pUL51 to hot-wire the ESCRT machinery. Previous studies have shown that these two proteins bind to each other, but it remained unclear how they work. Butt et al. used a combination of biochemical and biophysical techniques to solve the three-dimensional structures of pUL7 and pUL51 when bound to each other. The experiments determined that the structure of pUL51 resembles the structures of different components in the ESCRT machinery. Like the ESCRT proteins, pUL51 formed filaments, suggesting that pUL51 bends membranes in cells and that pUL7 blocks it from doing so until the time is right. Further experiments showed that the equivalents of pUL7 and pUL51 in other members of the herpes virus family also bind to each other in a similar way. These findings reveal that herpes simplex viruses and their close relatives have evolved a different strategy than many other viruses to steal membranes from host cells. Interfering with this mechanism may provide new avenues for designing drugs or improving vaccines against these viruses. The pUL7 and pUL51 proteins may also inspire new tools in biotechnology that could precisely control the shapes of biological membranes.

Mannose Binding Lectin Is Hydroxylated by Collagen Prolyl-4-hydroxylase and Inhibited by Some PHD Inhibitors.

Background: Mannose-binding lectin (MBL) is an important component of innate immune defense. MBL undergoes oligomerization to generate high mol weight (HMW) forms which act as pattern recognition molecules to detect and opsonize various microorganisms. Several post-translational modifications including prolyl hydroxylation are known to affect the oligomerization of MBL. Yet, the enzyme(s) which hydroxylate proline in the collagen-like domain residues have not been identified and the significance of prolyl hydroxylation is incompletely understood. Methods: To investigate post-translational modifications of MBL, we stably expressed Myc-DDK tagged MBL in HEK293S cells. We used pharmacologic and genetic inhibition of 2-oxoglutarate-dependent dioxygenases (2OGDD) to identify the enzyme required for prolyl hydroxylation of MBL. We performed mass spectrometry to determine the effects of various inhibitors on MBL modifications. Results: Secretion of HMW MBL was impaired by inhibitors of the superfamily of 2OGDD, and was dependent on prolyl-4-hydroxylase subunit α1. Roxadustat and vadadustat, but not molidustat, led to significant suppression of hydroxylation and secretion of HMW forms of MBL. Conclusions: These data suggest that prolyl hydroxylation in the collagen-like domain of MBL is mediated by collagen prolyl-4-hydroxylase. Reduced MBL activity is likely to be an off-target effect of some, but not all, prolyl hydroxylase domain (PHD) inhibitors. There may be advantages in selective PHD inhibitors that would not interfere with MBL production.