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.
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•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.
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.
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•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.
GeoBioMed - a new transdisciplinary approach that integrates the fields of geology, biology and medicine - reveals that kidney stones composed of calcium-rich minerals precipitate from a continuum of ...repeated events of crystallization, dissolution and recrystallization that result from the same fundamental natural processes that have governed billions of years of biomineralization on Earth. This contextual change in our understanding of renal stone formation opens fundamentally new avenues of human kidney stone investigation that include analyses of crystalline structure and stratigraphy, diagenetic phase transitions, and paragenetic sequences across broad length scales from hundreds of nanometres to centimetres (five Powers of 10). This paradigm shift has also enabled the development of a new kidney stone classification scheme according to thermodynamic energetics and crystalline architecture. Evidence suggests that ≥50% of the total volume of individual stones have undergone repeated in vivo dissolution and recrystallization. Amorphous calcium phosphate and hydroxyapatite spherules coalesce to form planar concentric zoning and sector zones that indicate disequilibrium precipitation. In addition, calcium oxalate dihydrate and calcium oxalate monohydrate crystal aggregates exhibit high-frequency organic-matter-rich and mineral-rich nanolayering that is orders of magnitude higher than layering observed in analogous coral reef, Roman aqueduct, cave, deep subsurface and hot-spring deposits. This higher frequency nanolayering represents the unique microenvironment of the kidney in which potent crystallization promoters and inhibitors are working in opposition. These GeoBioMed insights identify previously unexplored strategies for development and testing of new clinical therapies for the prevention and treatment of kidney stones.
HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug ...resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR 15N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.
Background: HIV-1 protease is an essential enzyme for HIV maturation.
Results: Select and naturally occurring polymorphisms alter the conformational sampling and backbone dynamics of HIV-1 protease.
Conclusion: These mutations lead to an alternative flap ensemble that we suspect is a curled flap orientation.
Significance: The mechanism of distal mutations on drug resistance is unclear, but altered dynamics and conformational equilibria likely play key roles.
The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are ...attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed "curled/tucked", "closed", "semi-open" and "wide-open" conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.
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In eukaryotic cells, heat shock triggers the accumulation of proteins into stress granules marked by poly(A)‐binding protein, or Pab1 in yeast. Formation of stress granules has been ...reported to involve a liquid‐liquid phase separation via multivalent interactions promoted by intrinsically disordered regions (IDRs). Here we report that Pab1 autonomously phase separates into a protein‐rich gel in vitro in response to physiological perturbations such as heat and acid. Pab1's highly conserved IDR, the proline‐rich “P domain”, tunes but is not necessary for the phase separation in vivo and in vitro. Evolutionary analysis of the P domain reveals compositional selection for low charge (<10%), a unique selection on its aliphatic residues, and a conserved above‐average hydrophobicity compared to other folded and disordered domains. We find that the domain collapses but is disordered by SAXS, CD and NMR. Collapse is robust, retained upon sequence randomization or substitution of all glycines (14% of residues) to prolines (from 19% to 33%) or alanines (from 12% to 26%). Of the mutations tested, only those to aliphatics or aromatics that lessen the IDR's hydrophobicity perturb its collapse, in contrast to expectations from studies of polar tracts (ie polyQ). Additionally, while previous results suggested that phase separation involves IDR‐IDR interactions between polar, aromatic, and charged residues, we find that hydrophobic interactions from aliphatic residues in the P domain modulate phase separation in vivo and in vitro. Mutations that reduce Pab1's heat‐triggered phase separation also reduce cells' ability to grow at during heat stress, indicating that phase separation is adaptive. Our results indicate that Pab1's heat‐induced phase separation represents a largely autonomous, evolutionarily tuned self‐assembly response to stress.
The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are ...attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed "curled/tucked", "closed", "semi-open" and "wide-open" conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.
The conformational landscape of HIV-1 protease can be characterized by double electron-electron resonance (DEER) spin-labeling.
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