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Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novelin uterostable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)–based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress., P. L. -M. was funded by a PhD fellowship from the ‘‘la Caixa’’ Foundation and received financial support from the Universidad de Zaragoza, Fundación Bancaria Ibercaja, and Fundación CAI (CM 1/16) during his stay at the UW–Madison. We acknowledge Ministerio de Economía, Industria y Competitividad for grant AGL2015-66177 (J. A. C.), the University of Zaragoza for grant UZ-2015-BIO-01 (J. A. C.), and OTRI (University of Zaragoza), Ministerio de Ciencia, Innovación y Universidades for grants CTQ2013-44367-C2-2-P and BFU2016-75633-P (R. H. -G.) and BFU2016-78232-P (A. V. -C.), Gobierno de Aragón for grants E34_R17 and LMP58_18 (R. H. -G.) and E45_17R (J. A. C and A. V. -C.) and FEDER (2014–2020) funds for “Building Europe from Aragón”. R. H. -G. and A. V. -C. thank the ARAID foundation for support. The research leading to these results also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement N283570 and BIOSTRUCTX_5186). We also thank the ALBA synchrotron radiation source, in particular, the beamline XALOC facility. J. A. and I. D. acknowledge access to UEA Faculty of Science Research Facilities. J. A. acknowledges funding support from the Biotechnology and biological Sciences Research Council (BBSRC; BB/P010660/1) and the Spanish Ministry of Science, Innovation and Universities through grant PID2019-109395GB-100. T. H. acknowledges a BBSRC DTP studentship. Financial support from the Universidad de Sevilla (Acciones Especiales del VI Plan Propio de Investigación y Transferencia) is also acknowledged. This work was also supported by National Institutes of Health (NIH) Grant GM065386 (J. M. D.), NIH National Research Service Award T32 GM007215 (J. B.) and the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) DGE-1256259 (J. B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Stable isotope labeling by amino acids in cell culture (SILAC) coupled to data-dependent acquisition (DDA) is a common approach to quantitative proteomics with the desirable benefit of reducing batch effects during sample processing and data acquisition. More recently, using data-independent acquisition (DIA/SWATH) to systematically measure peptides has gained popularity for its comprehensiveness, reproducibility, and accuracy of quantification. The complementary advantages of these two techniques logically suggests combining them. Here, we develop a SILAC-DIA-MS workflow using free, open-source software. We determine empirically that using DIA achieves similar peptide detection numbers as DDA and that DIA improves the quantitative accuracy and precision of SILAC by an order of magnitude. Finally, we apply SILAC-DIA-MS to determine protein turnover rates of cells treated with bortezomib, a 26S proteasome inhibitor FDA-approved for multiple myeloma and mantle cell lymphoma. We observe that SILAC-DIA produces more sensitive protein turnover models. Of the proteins determined differentially degraded by both acquisition methods, we find known ubiquitin-proteasome degrands such as HNRNPK, EIF3A, and IF4A1/EIF4A-1, and a slower turnover for CATD, a protein implicated in invasive breast cancer. With improved quantification from DIA, we anticipate this workflow making SILAC-based experiments like protein turnover more sensitive.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, an increasing concern is the long-term health implications. We augmented our IVF mouse model to longitudinally investigate cardiometabolic outcomes in offspring from optimal neonatal litter sizes. We found that IVF-conceived females had higher body weight and cholesterol levels compared to naturally-conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through transcriptomics and proteomics of adult liver, we identified sexually-dimorphic dysregulation of the sterol regulatory element binding protein (SREBP) pathways that are associated with the sex-specfic phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and β-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain–focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain–containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of β-hemoglobinopathies.

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Protein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell; however, identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation is an important factor to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under progrowth conditions. Finally, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well described. Data sets can be accessed through ProteomExchange via the MassIVE repository (ProteomExchange: PXD014453; MassIVE: MSV000084029).

24 pags, 6 figs. -- Supplemental Information includes five figures, five tables, and four videos and can be found with this article online at https://doi.org/10.1016/j.molcel.2018.07.031., Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3β-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination., Recombinant SIRT1 and SIRT2 were kindly supplied by Beatriz Camacho (UW Madison) and Mark A. Klein (UW Madison), respectively. Jin-Hee Lee (UW Madison) provided the X. laevis Histone H4. Susana Llanos (CNIO, Madrid, Spain) provided the human p300-HA clone. We thank Jing Fan (UW Madison) for discussions on metabolite flux and tracing experiments. We thank S.V. Medaris (UW Madison) for providing graphical support for Rgure 7. P.L.-M. was funded by a predoctoral fellowship from the "la Caixa" Foundation and received financial support from the Universidad de Zaragoza, Fundacion Bancaria lbercaja y Fundacion CAI (CM 1/16) during his stay at the UW Madison. This work has been funded by grants DK100263 and AG028730 (to D.A.S.). We acknowledge grants AGL2015-66177 (to P.L.-B. and J.A.C.) and CTQ2015-67727-R (to F.C.) from the Ministerio de Economia. Industria y Competitividad and UZ 2014-CIE-03 (to P.L-B.) and UZ-2015-B10-01 (to J.A.C.) from the University of Zaragoza. We thank ARAID and MEC (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H.-G.). J.M.D acknowledges NIH grant GM065386.

Protein acetylation of lysine ε-amino groups is abundant in cells, particularly within mitochondria. The contribution of enzyme-catalyzed and nonenzymatic acetylation in mitochondria remains unresolved. Here, we utilize a newly developed approach to measure site-specific, nonenzymatic acetylation rates for 90 sites in eight native purified proteins. Lysine reactivity (as second-order rate constants) with acetyl-phosphate and acetyl-CoA ranged over 3 orders of magnitude, and higher chemical reactivity tracked with likelihood of dynamic modification in vivo, providing evidence that enzyme-catalyzed acylation might not be necessary to explain the prevalence of acetylation in mitochondria. Structural analysis revealed that many highly reactive sites exist within clusters of basic residues, whereas lysines that show low reactivity are engaged in strong attractive electrostatic interactions with acidic residues. Lysine clusters are predicted to be high-affinity substrates of mitochondrial deacetylase SIRT3 both in vitro and in vivo. Our analysis describing rate determination of lysine acetylation is directly applicable to investigate targeted and proteome-wide acetylation, whether or not the reaction is enzyme catalyzed.

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD+-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ϵ-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6. Background: Sirtuins regulate metabolism, genome maintenance, and stress responses. Results: Long-chain free fatty acids stimulate SIRT6 deacetylase, and sirtuins display distinct but overlapping specificity for diverse acylated peptides. Conclusion: SIRT6 is activated by biologically relevant fatty acids, and long-chain deacylation is a general feature of sirtuins. Significance: Discovery of endogenous, small-molecule activators of SIRT6 demonstrates the therapeutic potential of compounds that promote SIRT6 function.

Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated.

Reversible protein acetylation is a major regulatory mechanism for controlling protein function. Through genetic manipulations, dietary perturbations, and new proteomic technologies, the diverse functions of protein acetylation are coming into focus. Protein acetylation in mitochondria has taken center stage, revealing that 63% of mitochondrially localized proteins contain lysine acetylation sites. Here we summarize the field, and discuss salient topics that cover spurious versus targeted acetylation, the role of SIRT3 deacetylation, nonenzymatic acetylation, and molecular models for regulatory acetylations that display high and low stoichiometry.

Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

HIV co-infection significantly impacts the natural history of hepatitis C virus (HCV) by increasing plasma HCV viral load, accelerating liver disease progression, and reducing rates of HCV clearance. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the impact of HIV on cytokine expression is unknown. In this study, an HCV continuous infection cell culture system was modified to permit co-infection with HIV to test the hypothesis that virus-induced disregulation of immune-response genes, particularly interferons and TGF-β, may create a permissive environment for the initial establishment of HIV/HCV co-infection in the host. CCR5-expressing Huh-7.5 hepatoma cells were transduced with human CD4 antigen to allow HIV infection in vitro. Co-infection of CD4⁺ Huh-7.5 cells with HIV and HCV or co-culture of HIV-infected CD4⁺ Huh-7.5 cells and HCV-infected Huh-7.5 cells increased the level of HCV RNA compared to HCV mono-infection. Quantitative gene expression analysis revealed HIV-induced up regulation of most tested IFN family genes when compared to HCV or co-infection. HCV infection induced up regulation of many TGF family genes that were subsequently down-regulated in the presence of HIV or HIV/HCV. Interestingly, co-infection resulted in down regulation of several IFN genes and significant up regulation of TGF-β genes leading to an overall enhancement of HCV replication. These data suggest that HIV infection may influence HCV replication in vitro by increasing levels of HCV RNA, possibly through the differential regulation of endogenous IFN and TGF family genes.

This article describes Bacteria ID Chips ('BacChips'): an inexpensive, portable, and autonomous microfluidic platform for identifying pathogenic strains of bacteria. BacChips consist of a set of microchambers and channels molded in the elastomeric polymer, poly(dimethylsiloxane) (PDMS). Each microchamber is preloaded with mono-, di-, or trisaccharides and dried. Pressing the layer of PDMS into contact with a glass coverslip forms the device; the footprint of the device in this article is ∼6 cm(2). After assembly, BacChips are degased under large negative pressure and are stored in vacuum-sealed plastic bags. To use the device, the bag is opened, a sample containing bacteria is introduced at the inlet of the device, and the degased PDMS draws the sample into the central channel and chambers. After the liquid at the inlet is consumed, air is drawn into the BacChip via the inlet and provides a physical barrier that separates the liquid samples in adjacent microchambers. A pH indicator is admixed with the samples prior to their loading, enabling the metabolism of the dissolved saccharides in the microchambers to be visualized. Importantly, BacChips operate without external equipment or instruments. By visually detecting the growth of bacteria using ambient light after ∼4 h, we demonstrate that BacChips with ten microchambers containing different saccharides can reproducibly detect the ESKAPE panel of pathogens, including strains of: Enterococcus faecalis, Enteroccocus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterobacter cloacae. This article describes a BacChip for point-of-care detection of ESKAPE pathogens and a starting point for designing multiplexed assays that identify bacterial strains from clinical samples and simultaneously determine their susceptibility to antibiotics.