Novel histone modifications in microglia derived from a mouse model of chronic pain.

As the resident immune cells in the central nervous system, microglia play an important role in the maintenance of its homeostasis. Dysregulation of microglia has been associated with the development and maintenance of chronic pain. However, the relevant molecular pathways remain poorly defined. In this study, we used a mass spectrometry-based proteomic approach to screen potential changes of histone protein modifications in microglia isolated from the brain of control and cisplatin-induced neuropathic pain adult C57BL/6J male mice. We identified several novel microglial histone modifications associated with pain, including statistically significantly decreased histone H3.1 lysine 27 mono-methylation (H3.1K27me1, 54.8% of control) and H3 lysine 56 tri-methylation (7.5% of control), as well as a trend suggesting increased H3 tyrosine 41 nitration. We further investigated the functional role of H3.1K27me1 and found that treatment of cultured microglial cells for 4 consecutive days with 1-10 µM of NCDM-64, a potent and selective inhibitor of lysine demethylase 7A, an enzyme responsible for the demethylation of H3K27me1, dose-dependently elevated its levels with a greater than a two-fold increase observed at 10 µM compared to vehicle-treated control cells. Moreover, pretreatment of mice with NCDM-64 (10 or 25 mg/kg/day, i.p.) prior to cisplatin treatment prevented the development of neuropathic pain in mice. The identification of specific chromatin marks in microglia associated with chronic pain may yield critical insight into the contribution of microglia to the development and maintenance of pain, and opens new avenues for the development of novel nonopioid therapeutics for the effective management of chronic pain.

Enhancement of Proteome Coverage by Ion Mobility Fractionation Coupled to PASEF on a TIMS-QTOF Instrument.

Trapped ion-mobility spectrometry (TIMS) was used to fractionate ions in the gas phase based on their ion mobility (V s/cm2), followed by parallel accumulation-serial fragmentation (PASEF) using a quadrupole time-of-flight instrument to determine the effect on the depth of proteome coverage. TIMS fractionation (up to four gas-phase fractions) coupled to data-dependent acquisition (DDA)-PASEF resulted in the detection of ∼7000 proteins and over 70,000 peptides overall from 200 ng of human (HeLa) cell lysate per injection using a commercial 25 cm ultra high performance liquid chromatography (UHPLC) column with a 90 min gradient. This result corresponded to ∼19 and 30% increases in protein and peptide identifications, respectively, when compared to a default, single-range TIMS DDA-PASEF analysis. Quantitation precision was not affected by TIMS fractionation as demonstrated by the average and median coefficient of variation values that were less than 4% upon label-free quantitation of technical replicates. TIMS fractionation was utilized to generate a DDA-based spectral library for downstream data-independent acquisition (DIA) analysis of lower sample input using a shorter LC gradient. The TIMS-fractionated library, consisting of over 7600 proteins and 82,000 peptides, enabled the identification of ∼4000 and 6600 proteins from 10 and 200 ng of human (HeLa) cell lysate input, respectively, with a 20 min gradient, single-shot DIA analysis. Data are available in ProteomeXchange: identifier PXD033129.

Polyphenol Supplementation Reverses Age-Related Changes in Microglial Signaling Cascades.

Microglial activity in the aging neuroimmune system is a central player in aging-related dysfunction. Aging alters microglial function via shifts in protein signaling cascades. These shifts can propagate neurodegenerative pathology. Therapeutics require a multifaceted approach to understand and address the stochastic nature of this process. Polyphenols offer one such means of rectifying age-related decline. Our group used mass spectrometry (MS) analysis to explicate the complex nature of these aging microglial pathways. In our first experiment, we compared primary microglia isolated from young and aged rats and identified 197 significantly differentially expressed proteins between these groups. Then, we performed bioinformatic analysis to explore differences in canonical signaling cascades related to microglial homeostasis and function with age. In a second experiment, we investigated changes to these pathways in aged animals after 30-day dietary supplementation with NT-020, which is a blend of polyphenols. We identified 144 differentially expressed proteins between the NT-020 group and the control diet group via MS analysis. Bioinformatic analysis predicted an NT-020 driven reversal in the upregulation of age-related canonical pathways that control inflammation, cellular metabolism, and proteostasis. Our results highlight salient aspects of microglial aging at the level of protein interactions and demonstrate a potential role of polyphenols as therapeutics for age-associated dysfunction.

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

Chronic Voluntary Binge Ethanol Consumption Causes Sex-Specific Differences in Microglial Signaling Pathways and Withdrawal-associated Behaviors in Mice.

BACKGROUND: Microglia are the resident immune cells in the brain where they play essential roles in the development and maintenance of physiological functions of this organ. Aberrant activation of microglia is speculated to be involved in the pathogenesis of a variety of neurological disorders, including alcohol use disorders. Repeated binge ethanol (EtOH) consumption can have a profound impact on the function and integrity of the brain resulting in changes in behaviors such as withdrawal and reward. However, the microglial molecular and cellular pathways associated with EtOH binge consumption remain poorly understood. METHOD: In this study, adult C57BL/6J male and female mice were subjected daily to a gelatin-based drinking-in-the-dark voluntary EtOH consumption paradigm (3 h/d for 4 months) to characterize EtOH consumption and withdrawal-associated and anxiety-like behaviors. Brain microglia were isolated at the end and analyzed for protein expression profile changes using unbiased mass spectrometry-based proteomic analysis. RESULTS: Both male and female mice consistently consumed binge quantities of EtOH daily, resulting in blood EtOH levels > 80 mg/dl measured at the end of the 3-hour daily consumption period. Although female mice consumed a significantly greater amount of EtOH than male mice, EtOH withdrawal-associated anxiety-like behaviors measured by marble-burying, light-dark box, and elevated plus maze tests were predominantly observed in male mice. Proteomic analysis of microglia isolated from the brains of animals at the end of the 4-month binge EtOH consumption identified 117 and 37 proteins that were significantly up- or downregulated in EtOH-exposed male and female mice, respectively, compared to their pair-fed controls. Protein expression profile-based pathway analysis identified several cellular pathways that may underlie the sex-specific and EtOH withdrawal-associated behavioral abnormalities. CONCLUSION: Taken together, our findings revealed sex-specific changes in EtOH withdrawal-associated behaviors and signaling pathways in the mouse brain microglia and may help advance our understanding of the molecular, cellular, and behavioral changes related to human binge EtOH consumption.

Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model.

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2-h gradient on a 75-cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label-free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.

BMI1-UBR5 axis regulates transcriptional repression at damaged chromatin.

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a key role in maintaining epigenetic silencing during development. BMI1 also participates in gene silencing during DNA damage response, but the precise downstream function of BMI1 in gene silencing is unclear. Here we identified the UBR5 E3 ligase as a downstream factor of BMI1. We found that UBR5 forms damage-inducible nuclear foci in a manner dependent on the PRC1 components BMI1, RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is repressed at UV-induced lesions on chromatin, depletion of the PRC1 members or UBR5 alone derepressed transcription elongation at these sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the UV-induced lesions along with SPT16. We show that UBR5 ubiquitinates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 repress SPT16 enrichment at the damaged sites. Consistently, depletion of the FACT components effectively reversed the transcriptional derepression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and BMI1 KO cells are hypersensitive to UV, which supports the notion that faulty RNA synthesis at damaged sites is harmful to the cell fitness. Altogether, these results suggest that BMI1 and UBR5 repress the polymerase II (Pol II)-mediated transcription at damaged sites, by negatively regulating the FACT-dependent Pol II elongation.

Proteomic Profiling of Iron-Treated Ovarian Cells Identifies AKT Activation that Modulates the CLEAR Network.

Although iron is essential for cell survival, dysregulated levels can contribute to cancer development or even cell death. The underlying mechanisms mediating these events remain unclear. Herein, proteomic alterations are assessed in iron-treated ovarian cell lines using reverse phase protein array (RPPA) technology and potential functional responses via ingenuity pathway analysis (IPA). Using these approaches, upregulation of pathways modulating organismal death with alterations in mTOR, MAPK, and AKT signaling in HEY ovarian cancer cells in contrast to T80 non-malignant ovarian cells is noted. Since modulation of cell death is mediated in part via microphthalmia-associated transcription factor (MiTF) family, which regulates lysosomal biogenesis and autophagosome formation by upregulating expression of coordinated lysosomal expression and regulation (CLEAR) network, expression changes in these factors in response to iron are investigated. Increased transcription factor EB (TFEB) in T80 (relative to HEY), accompanied by its nuclear translocation and increased CLEAR network gene expression with iron, is identified. Inhibition of AKT alters these responses in contrast to mTOR inhibition, which has little effect. Collectively, these findings support use of RPPA/IPA technology to predict functional responses to iron and further implicate AKT pathway and MiTF members in iron-induced cellular responses in ovarian cells.

In Vivo Metabolic Tracing Demonstrates the Site-Specific Contribution of Hepatic Ethanol Metabolism to Histone Acetylation.

BACKGROUND: Epigenetic dysregulation through ethanol (EtOH)-induced changes in DNA methylation and histone modifications has been implicated in several alcohol-related disorders such as alcoholic liver disease. EtOH metabolism in the liver results in the formation of acetate, a metabolite that can be converted to acetyl-CoA, which can then be used by histone acetyltransferases to acetylate lysine residues. EtOH metabolism in the liver can also indirectly influence lysine acetylation through NAD+ -dependent sirtuin activity that is altered due to increases in NADH. As a proof-of-concept study to determine the direct influence of hepatic EtOH metabolism on histone acetylation changes, we used heavy-labeled EtOH (13 C2 ) and mass spectrometry (MS) to site specifically characterize lysine acetylation on histone proteins. METHODS: Eight-week-old male C57BL/6J mice were gavaged using a bolus dose of either 13 C2 -labeled EtOH (5 g/kg) or maltose dextrin. Blood and livers were collected at 0, 4, and 24 hours followed by histone protein enrichment and derivatization using acid extraction and propionylation, respectively. Metabolic tracing and relative quantitation of acetylated histone proteins were performed using a hybrid quadrupole-orbitrap mass spectrometer. Data were analyzed using MaxQuant, Xcalibur Qual Browser, and the Bioconductor package "mzR." The contribution of EtOH to histone acetylation was quantified using the change in relative abundance of stable isotope incorporation in acetylated peptides detected by MS. RESULTS: Data show significant incorporation of the EtOH-derived 13 C2 -label into N-terminal lysine acetylation sites on histones H3 and H4 after 4 hours, with rapid turnover of labeled histone acetylation sites and return to endogenous levels at 24 hours postgavage. Moreover, site-specific selectivity was observed in regard to label incorporation into certain lysine acetylation sites as determined by tandem mass spectrometry and comparison to isotope simulations. CONCLUSIONS: These data provide the first quantitative evidence of how hepatic EtOH metabolism directly influences histone lysine acetylation in a site-specific manner and may influence EtOH-induced gene expression through these transcriptionally activating chromatin marks.

Ischemia-responsive protein 94 is a key mediator of ischemic neuronal injury-induced microglial activation.

Neuroinflammation, especially activation of microglia, the key immune cells in the brain, has been proposed to contribute to the pathogenesis of ischemic stroke. However, the dynamics and the potential mediators of microglial activation following ischemic neuronal injury are not well understood. In this study, using oxygen/glucose deprivation and reoxygenation with neuronal and microglial cell cultures as an in vitro model of ischemic neuronal injury, we set out to identify neuronal factors released from injured neurons that are capable of inducing microglial activation. Conditioned media (CM) from hippocampal and cortical neurons exposed to oxygen/glucose deprivation and reoxygenation induced significant activation of microglial cells as well as primary microglia, evidenced by up-regulation of inducible nitric oxide synthase, increased production of nitrite and reactive oxygen species, and increased expression of microglial markers. Mechanistically, neuronal ischemia-responsive protein 94 (Irp94) was a key contributor to microglial activation since significant increase in Irp94 was detected in the neuronal CM following ischemic insult and immunodepletion of Irp94 rendered ischemic neuronal CM ineffective in inducing microglial activation. Ischemic insult-augmented oxidative stress was a major facilitator of neuronal Irp94 release, and pharmacological inhibition of NADPH oxidase significantly reduced the ischemic injury-induced neuronal reactive oxygen species production and Irp94 release. Taken together, these results indicate that neuronal Irp94 may play a pivotal role in the propagation of ischemic neuronal damage. Continued studies may help identify Irp94 and/or related proteins as potential therapeutic targets and/or diagnostic/prognostic biomarkers for managing ischemia-associated brain disorders.

Proteomic anaysis of aged microglia: shifts in transcription, bioenergetics, and nutrient response.

BACKGROUND: Age is the primary risk factor for many diseases. As such, age is a critical co-factor for examination in order to understand the progression and potential intervention in disease progression. Studies examining both the phenotype and transcriptome of aged microglia demonstrated a propensity for the development of a pro-inflammatory phenotype. Less well studied is the concomitant blunting of anti-inflammatory aspects of microglial function with age which also impact plasticity and repair in the CNS. METHODS: This study utilizes mass spectrometry-based proteomics to compare primary microglia from young and aged animals. RESULTS: This study revealed alterations in three clusters of inter-related proteins. The three pathways were inflammatory signaling, mitochondrial function, and cellular metabolism. Analysis of these clusters identified the protein rapamycin-insensitive companion of mTOR (RICTOR), a component of the mTORC2 complex, as a novel upstream regulator of several biological functions that are altered with age and potentially linked to phenotype development. A decrease in mTORC2-dependent AKT S473 phosphorylation, as assessed by insulin growth factor (IGF) treatment, was observed in aged microglia. This novel finding was confirmed by genetic manipulation of the microglial cell line. BV2 cells with diminished RICTOR displayed a phenotype that was strikingly similar to that of aged microglia. This finding is particularly relevant as the mTOR pathway already has a number of pharmacological modulators used clinically. CONCLUSIONS: The results suggest that microglia from aged mice show changes in cellular metabolism and energy regulation that might underlie the alterations in inflammatory signaling. Modulation of one pathway identified in our bioinformatic analysis, RICTOR, may provide an avenue by which deleterious aspects of the aging microglia can be attenuated. If successful, this could mean potentially delaying or diminishing the progress of diseases for which progressive inflammation is involved.

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

Characterization of a SILAC method for proteomic analysis of primary rat microglia.

Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function and degeneration of the central nervous system. Application of SILAC-based proteomic analysis would greatly facilitate the identification of cellular pathways regulating the multifaceted phenotypes of microglia. We and others have successfully SILAC-labeled immortalized murine microglial cell lines in previous studies. In this study, we report the development and evaluation of a SILAC-labeled primary rat microglia model. Although the isotope labeling scheme for primary microglia is drastically different from that of immortalized cell lines, our de novo and uninterrupted primary culture labeling protocol (DUP-SILAC) resulted in sufficient incorporation of SILAC labels for mass spectrometry-based proteomic profiling. In addition, label incorporation did not alter their morphology and response to endotoxin stimulation. Proteomic analysis of the endotoxin-stimulated SILAC-labeled primary microglia identified expected as well as potentially novel activation markers and pro-inflammatory pathways that could be quantified in a more physiologically relevant cellular model system compared to immortalized cell lines. The establishment of primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways under various physiological and pathological conditions. Proteomic MS data are available via ProteomeXchange with identifier PXD002759.

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement.

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry.

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.

Cytosolic proteins lose solubility as amyloid deposits in a transgenic mouse model of Alzheimer-type amyloidosis.

The extracellular accumulation of ß-amyloid peptide is a key trigger in the pathogenesis of Alzheimer's disease (AD). In humans, amyloid deposition precedes the appearance of intracellular inclusion pathology formed by cytosolic proteins such as Tau, α-synuclein and TDP-43. These secondary pathologies have not been observed in mice that model Alzheimer-type amyloidosis by expressing mutant amyloid precursor protein, with or without mutant presenilin 1. The lack of secondary pathology in these models has made it difficult to establish how amyloid deposition initiates the cascade of events that leads to secondary intracellular pathology that characterizes human AD. In transgenic mice that model Alzheimer-type amyloidosis, we sought to determine whether there is evidence of altered cytosolic protein folding by assessing whether amyloid deposition causes normally soluble proteins to misfold. Using a method that involved detergent extraction and sedimentation coupled with proteomic approaches, we identified numerous cytosolic proteins that show specific losses in solubility as amyloid accumulates. The proteins identified included glycolytic enzymes and members of the 14-3-3 chaperone family. A substantial accumulation of lysine 48-linked polyubiquitin was also detected. Overall, the data demonstrate that the accumulation of amyloid by some manner causes the loss of solubility intracellular cytosolic proteins.

Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.

BACKGROUND & AIMS: Sirtuin (SIRT1) is a nicotinamide adenine dinucleotide-dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors. Ethanol exposure disrupts SIRT1 activity and contributes to alcoholic liver disease in rodents, but the exact pathogenic mechanism is not clear. We compared mice with liver-specific deletion of Sirt1 (Sirt1LKO) mice with their LOX littermates (controls). METHODS: We induced alcoholic liver injury in male Sirt1LKO and control mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Liver and serum samples were collected. We also measured messenger RNA levels of SIRT1, SFRS10, and lipin-1ß and lipin-1α in liver samples from patients with alcoholic hepatitis and individuals without alcoholic hepatitis (controls). RESULTS: On the ethanol-containing diet, livers of Sirt1LKO mice accumulated larger amounts of hepatic lipid and expressed higher levels of inflammatory cytokines than control mice; serum of Sirt1LKO mice had increased levels of alanine aminotransferase and aspartate aminotransferase. Hepatic deletion of SIRT1 exacerbated ethanol-mediated defects in lipid metabolism, mainly by altering the function of lipin-1, a transcriptional regulator of lipid metabolism. In cultured mouse AML-12 hepatocytes, transgenic expression of SIRT1 prevented fat accumulation in response to ethanol exposure, largely by reversing the aberrations in lipin-1 signaling induced by ethanol. Liver samples from patients with alcoholic hepatitis had reduced levels of SIRT1 and a higher ratio of Lpin1ß/α messenger RNAs than controls. CONCLUSIONS: In mice, hepatic deletion of Sirt1 promotes steatosis, inflammation, and fibrosis in response to ethanol challenge. Ethanol-mediated impairment of hepatic SIRT1 signaling via lipin-1 contributes to development of alcoholic steatosis and inflammation. Reagents designed to increase SIRT1 regulation of lipin-1 can be developed to treat patients with alcoholic fatty liver disease.

The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents.

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis.

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO-), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z=181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.