Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging

Sirtuins are NAD⁺‐dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6‐ and SIRT7‐interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co‐immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 ( http://proteomecentral.proteomexchange.org/dataset/PXD000159 , http://proteomecentral.proteomexchange.org/dataset/PXD000850 ).     

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1^?/? mice. The lack of deleterious phenotypes in Surf1^?/? mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1^?/? vs. Surf1^+/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1^+/+ and Surf1^?/? mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1^+/+ and Surf1^?/? mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1^?/? adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1^?/? mice. Finally, mitochondrial unfolded protein response (UPR^mt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPR^mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1^?/? mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.     

Myostatin deficiency but not anti‐myostatin blockade induces marked proteomic changes in mouse skeletal muscle

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post‐developmental Mstn blockade. Using high‐resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn^?/?) and mice treated for 2‐weeks with REGN1033, an anti‐Mstn antibody. Relative to wild‐type animals, Mstn^?/? mice had a two‐fold greater muscle mass and a >1.5‐fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5‐fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn^?/? mice corroborates the mutiple physiological changes including slow‐to‐fast fiber type switch. Thus, the proteome‐wide protein expression differs between Mstn^?/? mice and mice subjected to specific Mstn blockade post‐developmentally, providing molecular‐level insights to inform mechanistic hypotheses to explain the observed functional differences.     

A role for LFA‐1 in delaying T‐lymphocyte egress from lymph nodes

Lymphocytes use the integrin leukocyte function‐associated antigen‐1 (LFA‐1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA‐1 dependent. We show that LFA‐1 mediates prolonged LN residence as LFA‐1^?/? CD4 T cells have significantly decreased dwell times compared with LFA‐1^+/+ T cells, a distinction lost in hosts lacking the major LFA‐1 ligand ICAM‐1. Intra‐vital two‐photon microscopy revealed that LFA‐1^+/+ and LFA‐1^?/? T cells reacted differently when probing the ICAM‐1‐expressing lymphatic network. While LFA‐1^+/+ T cells returned to the LN parenchyma with greater frequency, LFA‐1^?/? T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA‐1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T‐cell response to antigen. Thus, we identify a novel function for LFA‐1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.     

BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinase

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3^+/+ and Tgfbr3^?/? mouse embryos, we show that Tgfbr3^?/? epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3^?/? epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3^+/+ cells; BMP2 induces cellular migration to Tgfbr3^+/+ levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3^?/? migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3^?/? epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.     

Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3‐targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3‐dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3‐targeted sites to chemical acetylation in vitro and fasting‐induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low‐level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues.     

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP‐dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super‐SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon‐dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon‐associated proteins were identified by label‐free LC‐MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.     

Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus

Recent analysis of prokaryotic N^ε‐lysine‐acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N^ε‐lysine‐acetylated proteome of gram‐positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl‐lysine‐specific antibodies followed by LC‐MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.     

HCN1 subunits contribute to the kinetics of Ih in neonatal cortical plate neurons

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel constituting the molecular substrate of hyperpolarization‐activated current (I_h). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I_h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole‐cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1^?/? and control HCN1^+/+ mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN^+/+ mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1^?/? mice other isoforms (particularly HCN4) might be compensatory up‐regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I_h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I_h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I_h properties in individual cortical plate neurons. These properties are significantly changed in HCN1^?/?, either due to the lack of HCN1 itself or due to compensatory mechanisms. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 785–797, 2013     

ADAMTS5 deficiency in mice does not affect cardiac function

The aggrecanase ADAMTS5 (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 motifs, member 5) and the cleavage of its substrate versican have been implicated in the development of heart valves. Furthermore, ADAMTS5 deficiency was shown to protect against diet‐induced obesity, a known risk factor for cardiovascular disease. Therefore, in this study, we investigated the potential role of ADAMTS5 in cardiac function using ADAMTS5‐deficient (Adamts5^?/?) mice and their wild‐type (Adamts5^+/+) counterparts exposed to a standard‐fat or a high‐fat diet (HFD). Eight‐weeks‐old Adamts5^?/? and Adamts5^+/+ mice were exposed to each diet for 15 weeks. Cardiac function and electrophysiology were analyzed by transthoracic echocardiogram and electrocardiogram at the end of the study. Cleavage of versican, as detected by the appearance of the DPEEAE neo‐epitope on western blotting with protein extracts, was defective in the heart of HFD‐treated Adamts5^?/? as compared with Adamts5^+/+ mice. ADAMTS5 deficiency led to statistically significant increases in diastolic posterior wall thickness (0.94 ± 0.023 vs. 0.82 ± 0.036 mm; P = 0.0056) and left ventricle volume (47 ± 4.5 vs. 31 ± 2.5 μL; P = 0.0043) in comparison to Adamts5^+/+ mice, but only in animals on a HFD. Cardiac function parameters such as ejection fraction, fractional shortening, and stroke volume were unaffected by ADAMTS5 deficiency or diet. Electrocardiogram analysis revealed no ADAMTS5‐specific changes in either diet group. Thus, in the absence of ADAMTS5, cleavage of versican in the cardiac extracellular matrix is impaired, but cardiac function, even upon exposure to a HFD, is not markedly affected.     

Brain serotonin determines maternal behavior and offspring survival

Maternal care is an indispensable component of offspring survival and development in all mammals and necessary for reproductive success. Although brain areas regulating maternal behaviors are innervated by serotonergic afferents, very little is known about the role of this neurotransmitter in these behaviors. To evaluate the contribution of serotonin to maternal care, we used mice with a null mutation in the gene for tryptophan hydroxylase‐2 (TPH2), which results in a genetic depletion of brain serotonin, and tested them in a wide range of maternal behavior paradigms. We found that litters born to and reared by TPH2^?/? mothers showed decreased survival, lower weaning weights and increased cannibalization. In addition, TPH2^?/? mothers performed poorly in pup retrieval, huddling, nest construction and high‐arched back nursing. Aggression in TPH2^?/? dams was not triggered by lactation and was steadily high. Survival and weaning weight deficits of TPH2^?/? pups were rescued by cross‐fostering and in litters of mixed genotype (TPH2^?/? and TPH2^?/+). However, the maternal behaviors of TPH2^?/? dams did not improve when rearing either TPH2^+/+ pups or mixed‐genotype litters. In addition, TPH2^?/? pups significantly worsened the behavior of TPH2^+/+ dams with respect to cannibalism, weaning weight and latency to attack. Olfactory and auditory functions of TPH2^?/? females or anxiety‐like behaviors did not account for these maternal alterations as they were equal to their TPH2^+/+ counterparts. These findings illustrate a profound influence of brain serotonin on virtually all elements of maternal behavior and establish that TPH2^?/? pups can engender maladaptive mothering in dams of both genotypes.     

Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN^+/+ mesenchymal stem cells (MSCs) exhibited improved pro‐angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN^?/? MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN^?/? MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN^+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro‐angiogenic function of WS‐MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.     

A novel role for acid‐sensing ion channels in Pavlovian reward conditioning

Pavlovian fear conditioning has been shown to depend on acid‐sensing ion channel‐1A (ASIC1A); however, it is unknown whether conditioning to rewarding stimuli also depends on ASIC1A. Here, we tested the hypothesis that ASIC1A contributes to Pavlovian conditioning to a non‐drug reward. We found effects of ASIC1A disruption depended on the relationship between the conditional stimulus (CS) and the unconditional stimulus (US), which was varied between five experiments. In experiment 1, when the CS preceded the US signaling an upcoming reward, Asic1a^?/? mice exhibited a deficit in conditioning compared to Asic1a^+/+ mice. Alternatively, in experiment 2, when the CS coinitiated with the US and signaled immediate reward availability, the Asic1a^?/? mice exhibited an increase in conditioned responses compared to Asic1a^+/+ mice, which contrasted with the deficits in the first experiment. Furthermore, in experiments 3 and 4, when the CS partially overlapped in time with the US, or the CS was shortened and coinitiated with the US, the Asic1a^?/? mice did not differ from control mice. The contrasting outcomes were likely because of differences in conditioning because in experiment 5 neither the Asic1a^?/? nor Asic1a^+/+ mice acquired conditioned responses when the CS and US were explicitly unpaired. Taken together, these results suggest that the effects of ASIC1A disruption on reward conditioning depend on the temporal relationship between the CS and US. Furthermore, these results suggest that ASIC1A plays a critical, yet nuanced role in Pavlovian conditioning. More research will be needed to deconstruct the roles of ASIC1A in these fundamental forms of learning and memory.     

Loss of SIRT2 leads to axonal degeneration and locomotor disability associated with redox and energy imbalance

Sirtuin 2 (SIRT2) is a member of a family of NAD⁺‐dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle‐aged, 13‐month‐old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2^?/? mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis.     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.