The conserved role of protein restriction in aging and disease.

PURPOSE OF REVIEW: Dietary interventions are effective strategies for preventing disease and promoting health span. Many of the effects of dietary restriction are linked to amino acid and protein levels and their regulation of nutrient-signaling pathways. Thus, protein restriction is a promising therapeutic strategy for preventing aging-related diseases and extending life span. RECENT FINDINGS: Studies in yeast and flies have shown that amino acid restriction promotes longevity and protection. In rodents, protein restriction extends life span and alleviates detrimental aging phenotypes. Finally, clinical trials in middle-aged adults have demonstrated the role of a protein-restricted diet in promoting health span. Interestingly, the population over the age of 65 may not benefit from severe protein restriction potentially because of the increased physiological decline that leads to decreased amino acid absorption and altered protein synthesis. SUMMARY: Protein restriction can have profound effects on health and longevity, but excessive restriction is detrimental, particularly in the very old. The investigation of the mechanisms that modulate nutrient-sensing pathways is important to understand how regulation of protein intake can optimize health span and longevity.

Heat shock and caloric restriction have a synergistic effect on the heat shock response in a sir2.1-dependent manner in Caenorhabditis elegans.

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth.

Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross ß-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific ß-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the entire cascade of aggregation intermediates formed along each pathway.

CCAR-1 is a negative regulator of the heat-shock response in Caenorhabditis elegans.

Defects in protein quality control during aging are central to many human diseases, and strategies are needed to better understand mechanisms of controlling the quality of the proteome. The heat-shock response (HSR) is a conserved survival mechanism mediated by the transcription factor HSF1 which functions to maintain proteostasis. In mammalian cells, HSF1 is regulated by a variety of factors including the prolongevity factor SIRT1. SIRT1 promotes the DNA-bound state of HSF1 through deacetylation of the DNA-binding domain of HSF1, thereby enhancing the HSR. SIRT1 is also regulated by various factors, including negative regulation by the cell-cycle and apoptosis regulator CCAR2. CCAR2 negatively regulates the HSR, possibly through its inhibitory interaction with SIRT1. We were interested in studying conservation of the SIRT1/CCAR2 regulatory interaction in Caenorhabditis elegans, and in utilizing this model organism to observe the effects of modulating sirtuin activity on the HSR, longevity, and proteostasis. The HSR is highly conserved in C. elegans and is mediated by the HSF1 homolog, HSF-1. We have uncovered that negative regulation of the HSR by CCAR2 is conserved in C. elegans and is mediated by the CCAR2 ortholog, CCAR-1. This negative regulation requires the SIRT1 homolog SIR-2.1. In addition, knockdown of CCAR-1 via ccar-1 RNAi works through SIR-2.1 to enhance stress resistance, motility, longevity, and proteostasis. This work therefore highlights the benefits of enhancing sirtuin activity to promote the HSR at the level of the whole organism.

The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1.

Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long- and branched-chain fatty acids for subsequent ß-oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross-linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging-related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging-related diseases indicating that peroxisome maintenance is a critical component of 'healthy aging'. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age-dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction.

Aging and SKN-1-dependent Loss of 20S Proteasome Adaptation to Oxidative Stress in C. elegans.

Aging is marked by a collapse of protein homeostasis and deterioration of adaptive stress responses that often lead to disease. During aging, the induction of stress responses decline along with protein quality control. Here, we have shown that the ability to mount an adaptive response by pretreatment with minor oxidative stress is abrogated in aged Caenorhabditis elegans We have identified a defect in SKN-1 signaling sensitivity during aging and have also found an aging-related increase in basal proteasome expression and in vitro activity, however, adaptation of the 20S proteasome in response to stress is lost in old animals. Interestingly, increased activation of SKN-1 promotes stress resistance, but is unable to rescue declining adaptation during aging. Our data demonstrate that the aging-dependent decline in SKN-1 signaling negatively impacts adaptation of the 20S proteasome in response to acute oxidative stress.

Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators.

Stress Inducibility of SIRT1 and Its Role in Cytoprotection and Cancer.

Cells must continuously respond to stressful insults via the upregulation of cytoprotective pathways. The longevity factor and deacetylase SIRT1 plays a critical role in coordinating this cellular response to stress. SIRT1 activity and levels are regulated by cellular stressors, including metabolic, genotoxic, oxidative, and proteotoxic stress. As a stress sensor, SIRT1 impacts cell survival by deacetylating substrate proteins to drive the cell towards a cytoprotective pathway. Extreme stress conditions, however, can cause SIRT1 to lead cells down an apoptotic pathway instead. SIRT1 is frequently dysregulated in cancer cells and has been characterized to have a dual role as both an oncogene and a tumor suppressor, likely due to its pivotal function in regulating cytoprotection. Recently, the ability of SIRT1 to regulate HSF1-dependent induction of the heat shock response has highlighted another pathway through which SIRT1 can modulate cytoprotection. Activation of HSF1 results in the production of cytoprotective chaperones that can facilitate the transformed phenotype of cancer cells. In this review, we discuss the stress-dependent regulation of SIRT1. We highlight the role of SIRT1 in stress management and cytoprotection and emphasize SIRT1-dependent activation of HSF1 as a potential mechanism for cancer promotion.

The SIRT1 modulators AROS and DBC1 regulate HSF1 activity and the heat shock response.

The heat shock response, the cellular response to protein damaging stress, is critical in maintaining proteostasis. The heat shock response is regulated by the transcription factor HSF1, which is activated upon heat shock and other stresses to induce the expression of molecular chaperones. SIRT1 has previously been shown to activate HSF1 by deacetylating it, leading to increased DNA binding ability. We have investigated how the heat shock response may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD(+)/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators AROS and DBC1 have an impact on hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. Therefore, AROS and DBC1 are now two new targets available for therapeutic regulation of the heat shock response.

HSF transcription factor family, heat shock response, and protein intrinsic disorder.

Intrinsically disordered proteins are highly abundant in all kingdoms of life, and several protein functional classes, such as transcription factors, transcriptional regulators, hub and scaffold proteins, signaling proteins, and chaperones are especially enriched in intrinsic disorder. One of the unique cellular reactions to protein damaging stress is the so-called heat shock response that results in the upregulation of heat shock proteins including molecular chaperones. This molecular protective mechanism is conserved from prokaryotes to eukaryotes and allows an organism to respond to various proteotoxic stressors, such as heat shock, oxidative stress, exposure to heavy metals, and drugs. The heat shock response- related proteins can be expressed during normal conditions (e.g., during the cell growth and development) or can be induced by various pathological conditions, such as infection, inflammation, and protein conformation diseases. The initiation of the heat shock response is manifested by the activation of the heat shock transcription factors HSF 1, part of a family of related HSF transcription factors. This review analyzes the abundance and functional roles of intrinsic disorder in various heat shock transcription factors and clearly shows that the heat shock response requires HSF flexibility to be more efficient.