Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify ...heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.
Display omitted
•Mass spectrometry quantifies aggregation of endogenous proteins during heat stress•Aggregates form rapidly in specific subcellular compartments•Endogenous protein aggregates are disassembled without degradation during recovery•In vitro, a heat-aggregated enzyme complex retains activity and fidelity
The aggregates of endogenous proteins triggered by heat stress in yeast are reversible. Rather than representing irreparably misfolded proteins destined for degradation, they can maintain activity and re-solubilize, suggesting an adaptive strategy underlying aggregation.
Heat shock induces a conserved transcriptional program regulated by heat shock factor 1 (Hsf1) in eukaryotic cells. Activation of this heat shock response is triggered by heat-induced misfolding of ...newly synthesized polypeptides, and so has been thought to depend on ongoing protein synthesis. Here, using the budding yeast
, we report the discovery that Hsf1 can be robustly activated when protein synthesis is inhibited, so long as cells undergo cytosolic acidification. Heat shock has long been known to cause transient intracellular acidification which, for reasons which have remained unclear, is associated with increased stress resistance in eukaryotes. We demonstrate that acidification is required for heat shock response induction in translationally inhibited cells, and specifically affects Hsf1 activation. Physiological heat-triggered acidification also increases population fitness and promotes cell cycle reentry following heat shock. Our results uncover a previously unknown adaptive dimension of the well-studied eukaryotic heat shock response.
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other ...assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1’s LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Display omitted
•Physiological thermal and pH changes trigger demixing of poly(A)-binding protein•RNA inhibits demixing, and a low-complexity region (LCR) tunes but does not cause it•Mutations that impede stress-triggered demixing also reduce stress tolerance•Phase separation confers exceptional and adaptive thermal sensitivity
Demixing of an abundant RNA-binding protein into hydrogel droplets, triggered by stress-associated physiological conditions, promotes cell fitness during stress.
Mitochondria are an essential component of cellular integrity and homeostasis, and their functions and pathological processes are highly dependent on mitochondrial ion channels. Anion channels of the ...inner mitochondrial membrane have been described by direct patch‐clamp electrophysiological methods in mitoplasts prepared in cardiac, liver, and brown adipose tissue, but not in brain. Here, using acutely isolated rat brain mitoplasts, we describe the properties of a large conductance, voltage‐gated, pH‐sensitive, outwardly rectifying chloride channel with conductances of 98 pS and 129 pS at negative and positive membrane potentials, respectively. While the molecular identity of this chloride conductance is unknown, it is unlikely to be a CLIC channel due to differences in the observed electrophysiological properties.
Thermostable enzymes combine catalytic specificity with the toughness required to withstand industrial reaction conditions. Stabilized enzymes also provide robust starting points for evolutionary ...improvement of other protein properties. We recently created a library of at least 2,300 new active chimeras of the biotechnologically important cytochrome P450 enzymes. Here we show that a chimera's thermostability can be predicted from the additive contributions of its sequence fragments. Based on these predictions, we constructed a family of 44 novel thermostable P450s with half-lives of inactivation at 57 degrees C up to 108 times that of the most stable parent. Although they differ by as many as 99 amino acids from any known P450, the stable sequences are catalytically active. Among the novel functions they exhibit is the ability to produce drug metabolites. This chimeric P450 family provides a unique ensemble for biotechnological applications and for studying sequence-stability-function relationships.
UAVs for coastal surveying Turner, Ian L.; Harley, Mitchell D.; Drummond, Christopher D.
Coastal engineering (Amsterdam),
August 2016, 2016-08-00, Letnik:
114
Journal Article
Recenzirano
UAVs (Unmanned Aerial Vehicles or “drones”) for routine survey applications at the coast have come of age, and are no longer ‘the latest thing’ more suited to the specialist researcher or amateur ...enthusiast. Off-the-shelf, survey-grade UAV equipment, data processing and analysis tools are now readily available to practicing coastal engineers, managers and researchers. Within the regulatory constraints that determine their use in many countries, UAVs provide an efficient and cost-effective survey tool for topographic mapping and measurement in the coastal zone. At the practical level, the specialist training required to operate off-the-shelf UAV suited to coastal surveying is now comparable in time and degree of difficulty to learning how to use the equivalent survey capabilities of professional hand-held RTK-GPS equipment. While incremental improvements to both the flight technology and data processing will no doubt continue to occur, from the coastal practitioner's perspective, no more step changes in UAV technology or ease of useability are required. In particular, survey-grade UAVs that incorporate internal RTK-GPS for high accuracy positioning and requiring a single operator only to safely deploy in the field, remove the need for separate and time-consuming on-ground surveying of ground control points (GCPs), previously required during post-deployment data processing. A coastal engineering application of UAV is used here to exemplify the practical use and potential benefits of this now mature survey technology. Over the past 2years, rapid post-storm deployment of UAV surveying has been successfully integrated into an established coastal monitoring program spanning 4 decades at Narrabeen Beach, Australia. This has extended the scope of this program to include detailed measurements of dune and beachface erosion spanning the full 3.5km long embayment at a spatial scale and temporal resolution that were previously unfeasible. For both the researcher and practicing coastal engineer, UAVs now provide a practical option for routine coastal surveying.
•UAVs (or “drones”) provide a practical solution for routine surveying at the coast.•Off-the-shelf UAV data processing and analysis tools are now readily available.•UAV incorporating RTK positioning removes the need for additional GCP surveys.•UAV-RTK survey accuracy is comparable to on-ground RTK-GPS beach survey methods.
We evaluated culture-negative, community-acquired endocarditis by using indirect immunofluorescent assays and molecular analyses for Bartonella spp. and Coxiella burnetii and found a prevalence of ...19.6% and 7.8%, respectively. Our findings reinforce the need to study these organisms in patients with culture-negative, community-acquired endocarditis, especially B. henselae in cat owners.
Background:
The mechanisms underlying the clinical effects of CBD remain poorly understood. Given the increasing evidence for CBD's effects on mitochondria, we sought to examine in more detail ...whether CBD impacts mitochondrial function and neuronal integrity.
Methods:
We utilized BE(2)-M17 neuroblastoma cells or acutely isolated brain mitochondria from rodents using a Seahorse extracellular flux analyzer and a fluorescent spectrofluorophotometer assay. Mitochondrial ion channel activity and hippocampal long-term potentiation were measured using standard cellular electrophysiological methods. Spatial learning/memory function was evaluated using the Morris water maze task. Plasma concentrations of CBD were assessed with liquid chromatography–mass spectrometry, and cellular viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction neuronal injury assay.
Results:
At low micromolar concentrations, CBD reduced mitochondrial respiration, the threshold for mitochondrial permeability transition, and calcium uptake, blocked a novel mitochondrial chloride channel, and reduced the viability of hippocampal cells. These effects were paralleled by
in vitro
and
in vivo
learning/memory deficits. We further found that these effects were independent of cannabinoid receptor 1 and mitochondrial G-protein-coupled receptor 55.
Conclusion:
Our results provide evidence for concentration- and dose-dependent toxicological effects of CBD, findings that may bear potential relevance to clinical populations.
Abstract only
How do cells sense and respond to changes in the environment? During heat stress, numerous eukaryotic RNA and protein species assemble into macromolecular bodies ‐ stress granules ‐ the ...function of which is largely unknown. Simultaneously, cells increase transcription of genes encoding heat shock proteins, many of which are molecular chaperones. In addition to increased transcription, the mRNA for these genes is also translated more efficiently. Thus, heat shock protein production is regulated by both transcription and translation. These heat‐induced mRNAs have exceptionally A‐rich 5′ untranslated regions (UTRs), which have long been known to mediate cap‐independent translation. We observe that poly(A)‐binding protein (PABP, Pab1 in S. cerevisiae) is bound to these A‐rich UTRs during exponential growth. Pab1 is able to repress translation of its own mRNA by binding a similar A‐rich UTR. We find that Pab1 represses translation of mRNA by binding A‐rich UTRs of heat shock proteins. Further, we propose that during heat‐shock, as Pab1 assembles into heat‐induced RNA‐protein granules, Pab1 releases bound 5′ UTRs, and relieves repression. We have recapitulated Pab1‐mediated repression and heat‐induced relief with an in vitro translation system. Moreover, yeast expressing assembly‐impaired Pab1 are not able to grow at elevated temperature; this result is consistent with disruption of an assembly‐mediated regulatory mechanism. Viewed through this lens, formation of RNA‐protein granules is a regulatory response, whose functions include translational regulation.
Support or Funding Information
The molecular and cellular biology training grant T32 GM007183
PDF
