Differential role of cytosolic Hsp70s in longevity assurance and protein quality control.

70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1Δ ssa2Δ mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast.

Absence of mitochondrial translation control proteins extends life span by activating sirtuin-dependent silencing.

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.

Opposing roles of Ubp3-dependent deubiquitination regulate replicative life span and heat resistance.

The interplay between molecular chaperones, ubiquitin/deubiquitinating enzymes, and proteasomes is a critical element in protein homeostasis. Among these factors, the conserved deubiquitinase, Ubp3, has the interesting ability, when overproduced, to suppress the requirement for the major cytosolic Hsp70 chaperones. Here, we show that Ubp3 overproduction counteracts deficiency of Hsp70s by the removal of damaged proteins deposited in inclusion bodies (JUNQ) during both aging and heat stress. Consistent with this, Ubp3 destabilized, deubiquitinated, and diminished the toxicity of the JUNQ-associated misfolded protein Ubc9(ts) in a proteasome-dependent manner. In contrast, another misfolded model protein, ssCPY*, was stabilized by Ubp3-dependent deubiquitination demonstrating a dual role for Ubp3, saving or destroying aberrant protein species depending on the stage at which the damaged protein is committed for destruction. We present genetic evidence for the former of these activities being key to Ubp3-dependent suppression of heat sensitivity in Hsp70-deficient cells, whereas protein destruction suppresses accelerated aging. We discuss the data in view of how heat stress and aging might elicit differential damage and challenges on the protein homeostasis network.

Reversal of RNA polymerase II ubiquitylation by the ubiquitin protease Ubp3.

The final outcome of protein polyubiquitylation is often proteasome-mediated proteolysis, meaning that "proofreading" of ubiquitylation by ubiquitin proteases (UBPs) is crucial. Transcriptional arrest can trigger ubiquitin-mediated proteolysis of RNA polymerase II (RNAPII) so a UBP reversing RNAPII ubiquitylation might be expected. Here, we show that Ubp3 deubiquitylates RNAPII in yeast. Genetic characterization of ubp3 cells is consistent with a role in elongation, and Ubp3 can be purified with RNAPII, Def1, and the elongation factors Spt5 and TFIIF. This Ubp3 complex deubiquitylates both mono- and polyubiquitylated RNAPII in vitro, and ubp3 cells have elevated levels of ubiquitylated RNAPII in vivo. Moreover, RNAPII is degraded faster in a ubp3 mutant after UV irradiation. Problems posed by damage-arrested RNAPII are thought to be resolved either by removing the damage or degrading the polymerase. In agreement with this, cells with compromised DNA repair are better equipped to survive UV damage when UPB3 is deleted.

Increased RNA polymerase availability directs resources towards growth at the expense of maintenance.

Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such 'passive' regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Esigma(70)) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress-defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Esigma(70) favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Esigma(70) compared with wild-type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Esigma(70). We conclude that ppGpp modulates the levels of free Esigma(70) and that this is an integral part of the alarmone's means of regulating a trade-off between growth and maintenance.

Teaching about antibiotic resistance to a broad audience: a multidisciplinary approach.

Education for the general public about antibiotic resistance is advocated as a key component of our response to this crisis. Since this is a multidisciplinary problem encompassing natural, medical and social sciences, it is an educational challenge as both students and lecturers will have vastly different backgrounds in the topics. Here we describe an online multidisciplinary course on antibiotic resistance spanning topics as diverse as chemistry and practical philosophy. The target group was any post-secondary school student and the participating students had different occupations and educational experience. Although as many as 38% of the students were currently studying natural sciences at university, the course included a diverse group with medical professionals (16%) and teachers (6%) making up a significant fraction of the class. The outcomes based on examination and the course evaluations were very positive and we have indications that the information students gained from this course has been spread to others. Unlike other online courses addressing antibiotic resistance, this course is both accessible to a wide range of students and covers a broad range of topics. We advocate courses like ours as an effective tool in educating the public about this crisis.

Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance.

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.1.

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.

Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae.

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

Regulation of sigma factor competition by the alarmone ppGpp.

Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.