Thomas Nyström

Role of oxidative carbonylation in protein quality control and senescence.

Proteins can become modified by a large number of reactions involving reactive oxygen species. Among these reactions, carbonylation has attracted a great deal of attention due to its irreversible and unrepairable nature. Carbonylated proteins are marked for proteolysis by the proteasome and the Lon protease but can escape degradation and form high‐molecular‐weight aggregates that accumulate with age. Such carbonylated aggregates can become cytotoxic and have been associated with a large number of age‐related disorders, including Parkinsons disease, Alzheimers disease, and cancer. This review focuses on the generation of and defence against protein carbonyls and speculates on the potential role of carbonylation in protein quality control, cellular deterioration, and senescence.

Unraveling the Biological Roles of Reactive Oxygen Species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

The bacterial universal stress protein: function and regulation.

The universal stress protein A (UspA) superfamily encompasses an ancient and conserved group of proteins that are found in bacteria, Archea, fungi, flies and plants. The Escherichia coli UspA is produced in response to a large number of different environmental onslaughts and UspA is one of the most abundant proteins in growth-arrested cells. Although insights into the regulation of the E. coli uspA gene have been gained, the exact roles of the Usp proteins and Usp domains remain enigmatic; they appear, in some cases, to be linked to resistance to DNA-damaging agents and to respiratory uncouplers.

The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates

The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome- and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2Delta cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice

The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate–nitrite–NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.

Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.

Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner.

Differential Roles of the Universal Stress Proteins of Escherichia coli in Oxidative Stress Resistance, Adhesion, and Motility

The universal stress protein (UspA) superfamily encompasses a conserved group of proteins that are found in bacteria, archaea, and eukaryotes. Escherichia coli harbors six usp genes--uspA, -C, -D, -E, -F, and -G--the expression of which is triggered by a large variety of environmental insults. The uspA gene is important for survival during cellular growth arrest, but the exact physiological role of the Usp proteins is not known. In this work we have performed phenotypic characterization of mutants with deletions of the six different usp genes. We report on hitherto unknown functions of these genes linked to motility, adhesion, and oxidative stress resistance, and we show that usp functions are both overlapping and distinct. Both UspA and UspD are required in the defense against superoxide-generating agents, and UspD appears also important in controlling intracellular levels of iron. In contrast, UspC is not involved in stress resistance or iron metabolism but is essential, like UspE, for cellular motility. Electron microscopy demonstrates that uspC and uspE mutants are devoid of flagella. In addition, the function of the uspC and uspE genes is linked to cell adhesion, measured as FimH-mediated agglutination of yeast cells. While the UspC and UspE proteins promote motility at the expense of adhesion, the UspF and UspG proteins exhibit the exact opposite effects. We suggest that the Usp proteins have evolved different physiological functions that reprogram the cell towards defense and escape during cellular stress.

Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells.

Analysis of protein carbonylation demonstrates that the stasis-induced catalases and cytoplasmic superoxide dismutases (SOD) have a role in preventing accelerated protein oxidation during growth arrest of Escherichia coli cells. A larger number of proteins are carbonylated in cells lacking cytoplasmic SOD compared with cells lacking catalases, OxyR, or RpoS which, in turn, exhibit a larger number of oxidized proteins than the wild-type parent. Proteins exclusively oxidized during stasis in mutants lacking cytoplasmic SOD include GroEL, EF-G, and the acidic isoform of H-NS indicating that these mutants experience problems in peptide elongation and maintaining protein and DNA architecture. These mutants also survive stasis poorly. Likewise, but to a much lesser extent, mutations in oxyR, an oxidative stress regulator, shorten the life-span of stationary phase cells. The low plating efficiency of cells lacking OxyR is the result of their inability to grow on standard culture plates unless plating is performed anaerobically or with high concentration of catalase. In contrast, cells lacking cytoplasmic SOD appear to die prior to plating. Our data points to the importance of oxidative stress defense in stasis survival, and we also demonstrate that the life-span of growth-arrested wild-type E. coli cells can be significantly extended by omitting oxygen.

Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition?

The regulatory design of higher organisms is proposed to comprise a trade‐off between activities devoted to reproduction and those devoted to cellular maintenance and repair. Excessive reproduction will inevitably limit the organisms ability to resist stress whereas excessively devoted stress defence systems may increase lifespan but reduce Darwinian fitness. The trade‐off is arguably a consequence of limited resources in any one organism but the nature and identity of such limiting resources are ambiguous. Analysis of global control of gene expression in Escherichia coli suggests that reproduction and maintenance activities are also at odds in bacteria and that this antagonism may be a consequence of a battle between transcription factors for limiting RNA polymerase. The outcome of this battle is regulated and depends on the nutritional status of the environment, the levels of the alarmone ppGpp, and RNA polymerase availability. This paper reviews how the concentration of RNA polymerase available for transcription initiation may vary upon shifts between growth and growth‐arrest conditions and how this adjustment may differentially affect genes whose functions relate to reproduction and maintenance.

Bacterial senescence: protein oxidation in non-proliferating cells is dictated by the accuracy of the ribosomes.

We have investigated the causal factors behind the age‐related oxidation of proteins during arrest of cell proliferation. A proteomic approach demonstrated that protein oxidation in non‐proliferating cells is observed primarily for proteins being produced in a number of aberrant isoforms. Also, these cells exhibited a reduced translational fidelity as demonstrated by both proteomic analysis and genetic measurements of nonsense suppression. Mutants harboring hyperaccurate ribosomes exhibited a drastically attenuated protein oxidation during growth arrest. In contrast, oxidation was augmented in mutants with error‐prone ribosomes. Oxidation increased concomitantly with a reduced rate of translation, indicating that the production of aberrant, and oxidized proteins, is not the result of titration of the co‐translational folding machinery. The age‐related accumulation of the chaperones, DnaK and GroEL, was drastically attenuated in the hyperaccurate rpsL mutant, demonstrating that the reduced translational fidelity in growth‐arrested cells may also be a primary cause for the induction of the heat shock regulon. The data point to an alternative way of approaching the causal factors involved in protein oxidation in eukaryotic G0 cells.