SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells.

Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.

SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including ß-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased ß-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

No consistent bioenergetic defects in presynaptic nerve terminals isolated from mouse models of Alzheimer's disease.

Depressed cortical energy supply and impaired synaptic function are predominant associations of Alzheimer's disease (AD). To test the hypothesis that presynaptic bioenergetic deficits are associated with the progression of AD pathogenesis, we compared bioenergetic variables of cortical and hippocampal presynaptic nerve terminals (synaptosomes) from commonly used mouse models with AD-like phenotypes (J20 age 6 months, Tg2576 age 16 months, and APP/PS age 9 and 14 months) to age-matched controls. No consistent bioenergetic deficiencies were detected in synaptosomes from the three models; only APP/PS cortical synaptosomes from 14-month-old mice showed an increase in respiration associated with proton leak. J20 mice were chosen for a highly stringent investigation of mitochondrial function and content. There were no significant differences in the quality of the synaptosomal preparations or the mitochondrial volume fraction. Furthermore, respiratory variables, calcium handling, and membrane potentials of synaptosomes from symptomatic J20 mice under calcium-imposed stress were not consistently impaired. The recovery of marker proteins during synaptosome preparation was the same, ruling out the possibility that the lack of functional bioenergetic defects in synaptosomes from J20 mice was due to the selective loss of damaged synaptosomes during sample preparation. Our results support the conclusion that the intrinsic bioenergetic capacities of presynaptic nerve terminals are maintained in these symptomatic AD mouse models.

Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms.

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.

Quantitative mapping of reversible mitochondrial Complex I cysteine oxidation in a Parkinson disease mouse model.

Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinson disease. We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within iron-sulfur clusters of Complex I, suggesting that their redox state may affect electron transport function.

Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.

Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease.

Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.

Oxidative and nitrative protein modifications in Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative syndrome likely involving contributions from various factors in individuals including genetic susceptibility, exposure to environmental toxins, and the aging process itself. Increased oxidative stress appears to be a common causative aspect involved in the preferential loss of dopaminergic neurons in a region of the brain prominently affected by the disorder, the substantia nigra (SN). Loss of dopaminergic SN neurons is responsible for the classic clinical motor symptoms associated with PD. Several oxidative and nitrative posttranslational modifications (PTMs) have been identified on proteins pertinent to PD that may affect this or other aspects of disease progression. In this review, we discuss several examples of such PTMs to illustrate their potential consequences in terms of initiation or progression of PD neuropathophysiology.

Isolation of transcriptomal changes attributable to LHON mutations and the cybridization process.

Leber's hereditary optic neuropathy (LHON) is thought to be the most common disease resulting from mitochondrial DNA (mtDNA) point mutations, and transmitochondrial cytoplasmic hybrid (cybrid) cell lines are the most frequently used model for understanding the pathogenesis of mitochondrial disorders. We have used oligonucleotide microarrays and a novel study design based on shared transcripts to allocate transcriptomal changes into rho-zero-dependent, cybridization-dependent and LHON-dependent categories in these cells. The analysis indicates that the rho-zero process has the largest transcriptomal impact, followed by the cybridization process, and finally the LHON mutations. The transcriptomal impacts of the rho-zero and cybridization processes preferentially and significantly affect the mitochondrial compartment, causing upregulation of many transcripts involved in oxidative phosphorylation, presumably in response to the mtDNA depletion that occurs at the rho-zero step. Nine LHON-specific transcriptional alterations were shared among osteosarcoma cybrids and lymphoblasts bearing LHON mutations. Notably, the aldose reductase transcript was overexpressed in LHON cybrids and lymphoblasts. Aldose reductase is also overexpressed in diabetic retinopathy, leading to optic nerve and retinal complications. The LHON-specific increase in transcript level was confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and a western blot confirmed a higher level of aldose reductase in mutant mitochondria. One product of aldose reductase is sorbitol, which has been linked to osmotic stress, oxidative stress and optic neuropathy, and sorbitol levels were increased in LHON cybrids. If these results are confirmed in patient tissues, aldose reductase inhibitors could have some therapeutic value for LHON.

Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-Induced apoptosis.

Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.