Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned 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.

Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress.

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.

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts.

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 role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics.

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 (15)N 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.

Human kidney stones: a natural record of universal biomineralization.

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.