Gel or Die: Phase Separation as a Survival Strategy.

Stress conditions trigger protein assembly by demixing from the cytoplasm, but the biological significance is still unclear. In this issue of Cell, Riback et al. report that the yeast poly(A)-binding protein 1 (Pab1) is a phase-separating stress sensor that boosts organismal fitness under physiological stress conditions.

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Imatinib mesylate and nilotinib decrease synthesis of bone matrix in vitro.

Tyrosine kinase inhibitors (TKIs), such as imatinib (IMA) and nilotinib (NIL), are the cornerstone of chronic myeloid leukemia (CML) treatment via the blockade of the oncogenic BCR-ABL1 fusion protein. However, skeletal side effects are commonly observed in pediatric patients receiving long-term treatment with IMA. Additionally, in vitro studies have shown that IMA and NIL alter vitamin D metabolism, which may further impair bone metabolism. To determine whether TKIs directly affect bone cell function, the present study treated the human osteoblastic cell line SaOS-2 with IMA or NIL and assessed effects on their mineralization capacity as well as mRNA expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), two cytokines that regulate osteoclastogenesis. Both TKIs significantly inhibited mineralization and downregulated osteoblast marker genes, including alkaline phosphatase, osteocalcin, osterix, as well as genes associated with the pro-osteogenic Wnt signaling pathway; NIL was more potent than IMA. In addition, both TKIs increased the RANKL/OPG ratio, which is known to stimulate osteoclastogenesis. The present results suggested that the TKIs IMA and NIL directly inhibited osteoblast differentiation and directly promoted a pro-osteoclastogenic environment through the RANKL-OPG signaling axis. Thus, we propose that future work is required to determine whether the bone health of CML patients undergoing TKI-treatment should be routinely monitored.

Different Material States of Pub1 Condensates Define Distinct Modes of Stress Adaptation and Recovery.

How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery.

Preliminary crystallographic characterization of PrnB, the second enzyme in the pyrrolnitrin biosynthetic pathway.

Pyrrolnitrin is the active ingredient of drugs for the treatment of superficial fungal infections and was used as a lead structure for the development of fludioxonil. It is an effective agent for plant diseases caused by the fungal pathogen Rhizoctonia solani. Pyrrolnitrin is made in four steps, the second of which, catalyzed by PrnB, is a novel chemical rearrangement of 7-chlorotryptophan. PrnB was overproduced in Pseudomonas fluorescens (BL915) and well diffracting crystals were obtained of a triple cysteine-to-serine mutant by sitting-drop vapour diffusion. Crystals grown in the presence of L-7-chlorotryptophan, D-tryptophan and L-tryptophan are reported. Data sets for each are reported with high-resolution limits of 2.0, 1.75 and 1.75 A, respectively. Two crystals (PrnB in the presence of D-tryptophan and L-7-chlorotryptophan) belong to space group C2 with similar unit-cell parameters (a = 68.6, b = 79.5, c = 92.7 A, alpha = gamma = 90.0, beta = 103.8 degrees). Crystals grown in the presence of L-tryptophan belong to space group C222(1) and have unit-cell parameters a = 67.7, b = 80.1, c = 129.5 A. All crystals contain a monomer in the asymmetric unit.

Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation.

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

Cell cortex composition and homeostasis resolved by integrating proteomics and quantitative imaging.

The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems-level investigations of the actomyosin cortex and its regulation during morphogenesis.

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.