Proteostasis and SUMO in the heart

Heart proteostasis relies on a complex and integrated network of molecular processes surveilling organ performance under physiological and pathological conditions. For this purpose, cardiac cells depend on the correct function of their proteolytic systems, such as the ubiquitin-proteasome system (UPS), autophagy and the calpain system. Recently, the role of protein SUMOylation (an ubiquitin-like modification), has emerged as important modulator of cardiac proteostasis, which will be the focus of this review.     

The Proteome Folding Problem and Cellular Proteostasis

Stunning advances have been achieved in addressing the protein folding problem, providing deeper understanding of the mechanisms by which proteins navigate energy landscapes to reach their native states and enabling powerful algorithms to connect sequence to structure. However, the realities of the in vivo protein folding problem remain a challenge to reckon with. Here, we discuss the concept of the “proteome folding problem”—the problem of how organisms build and maintain a functional proteome—by admitting that folding energy landscapes are characterized by many misfolded states and that cells must deploy a network of chaperones and degradation enzymes to minimize deleterious impacts of these off-pathway species. The resulting proteostasis network is an inextricable part of in vivo protein folding and must be understood in detail if we are to solve the proteome folding problem. We discuss how the development of computational models for the proteostasis network’s actions and the relationship to the biophysical properties of the proteome has begun to offer new insights and capabilities.     

Emergent Properties of Proteostasis in Managing Cystic Fibrosis

Cystic fibrosis (CF) is a consequence of defective recognition of the multimembrane spanning protein cystic fibrosis conductance transmembrane regulator (CFTR) by the protein homeostasis or proteostasis network (PN) ( Hutt and Balch (2010). Like many variant proteins triggering misfolding diseases, mutant CFTR has a complex folding and membrane trafficking itinerary that is managed by the PN to maintain proteome balance and this balance is disrupted in human disease. The biological pathways dictating the folding and function of CFTR in health and disease are being studied by numerous investigators, providing a unique opportunity to begin to understand and therapeutically address the role of the PN in disease onset, and its progression during aging. We discuss the general concept that therapeutic management of the emergent properties of the PN to control the energetics of CFTR folding biology may provide significant clinical benefit.     

Hedgehog Signaling Modulates Glial Proteostasis and Lifespan

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Interrelation Between Protein Synthesis,Proteostasis and Life Span

The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity.     

Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence

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Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals

Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole, Myodes glareolus. The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days⁻¹; liver 0.136 days⁻¹; heart, 0.054 days⁻¹, and skeletal muscle, 0.035 days⁻¹). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054.Proteostasis balances the opposing contributions of protein synthesis and protein degradation in the maintenance or adjustment of the intracellular abundance of a protein. Accurate determination of the net contribution of these two processes requires accurate determination of at least two of the three parameters of synthesis rate, degradation rate, and protein pool size. Moreover, these parameters need to be recoverable at proteome scales, extending to many proteins in a parallel analysis within a single experiment. Because synthesis and degradation can still occur even when the protein concentration is unchanging, it is necessary to monitor the flux through the protein pool by a tracer, and in a mass spectrometry-driven proteomics context, this involves the incorporation of a stable isotope label. The tracer can be administered as a metabolic precursor (1, 2), an amino acid (3–5), a microbially sourced diet uniformly labeled with ¹⁵N, or by administration of [²H₂]O in drinking water (6–9).Many studies of proteome dynamics have been conducted in mammalian cells in culture (for reviews see (10, 11)). The experimental convenience of effecting rapid switching between a labeled and a subsequent unlabeled precursor pool by medium exchange is, however, offset by the fact that rapidly dividing cells are able to “solve” the problem of protein level adjustment by dilution into progeny cells. This dilution of the isotopically labeled pool not only restricts the scope to monitor the labeled pool but also may be focusing turnover studies on cells that bear little resemblance in their protein turnover to the same or similar cells in a tissue (11, 12). There is therefore a requirement to determine protein turnover in intact animals, which imposes considerable complexity in experimental design and data analysis (13–16). In tissues, protein turnover is substantially slower than in cells in culture (12, 17), and it is necessary to administer label for extended periods, rendering oral (food or drinking water) administration as the only feasible option.Measurement of turnover in rapidly growing cells in culture suffers from the rapid loss or gain of label as a consequence of growth and, thus, dilution of the labeled protein pool. By contrast, measurement of turnover in animal tissues, particularly in nongrowing individuals, requires a different strategy (13, 14, 16, 18–20). It is challenging and prohibitively expensive to label animals fully over multiple generations and subsequently monitor the loss of label over time. Further, a strategy that measures the transition between fully labeled and fully unlabeled proteins requires specialist, completely labeled diets that differ substantially from normal laboratory diets. To circumvent such difficulties, we have developed a strategy based on the addition of a single stable isotope labeled amino acid to a laboratory diet, such that the isotopic enrichment of the total amino acid in the diet would be ∼0.5. The precise degree of dietary labeling is not critical, as this is revealed during analysis. Animals are acclimated to the modified diet containing nonlabeled amino acid before being transferred to the same composition, labeled diet. Subsequently, incorporation of label into the body precursor pool can be monitored noninvasively by measurement of the relative isotope abundance (RIA)¹ of secreted proteins, particularly those released in urine (16). We have previously used the essential amino acid, valine, to measure protein turnover in the house mouse, Mus musculus domesticus (16). The valine was administered by supplementation of a standard laboratory diet by incorporation of crystalline [²H₈]valine to the same level as originally present in the diet (in protein bound or free form). Thus, the dietary RIA for the valine was set to a nominal value of 0.5. This approach worked very well, apart from an additional (but surmountable, (16)) complication due to partial transamination of the valine that led to loss of the alpha carbon deuteron to form a mixture of [²H₈] and [²H₇]valine. In this study, to simplify the strategy for determination of protein turnover rates, we have substituted [¹³C₆]lysine for the [²H₈]valine used previously. Lysine is also an essential amino acid, and so the RIA of the precursor pool cannot be reduced by biosynthesis de novo. Moreover, the labeling at the six carbon atoms precludes complications due to metabolic loss of specific atom centers. Finally, approximately half of the tryptic peptides (those that are lysine-terminated or which contain an internal LysPro sequence) should yield informative turnover data, although all tryptic peptides (both lysine and arginine terminated) can of course be used for protein identification.We are interested in the allometric scaling of proteome turnover, which will require approaches that recover high-quality turnover rates from species for which fully annotated proteomes do not exist. To test the feasibility of this approach, we selected as our experimental system a rodent of similar body mass to the house mouse, the bank vole Myodes glareoulus. The annotated genome sequence of this rodent is not available, and thus, the measurement of protein turnover in this species brings two challenges: that of cross-species identification and also the recovery of species-specific turnover parameters. Finally, we have provided a robust analytical approach to the determination of protein turnover rates using the statistical package R that is thus amenable to use by all groups working in the field.     

Mechanical properties of the heart muscle in the passive state

The viscoelastic behaviour of the heart muscle (papillary muscle) in the passive unstimulated) state is studied by such methods as stress relaxation, creep, vibration and stress-strain testing. The tests are conducted on a newly developed electromechanical muscle testing device which is suitable for conducting active and passive tests on biological materials.     

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.