4 research outputs found
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Insights into Stress-Induced Condensation of mRNA and Protein
mRNA and protein clump—or condense—in response to cellular stress across the tree of eukaryotic life. Yet, despite decades of inquiry and its universal evolutionary conservation, the function of stress-induced condensation remains enigmatic. The aim of this thesis is to gain insights into this fundamental phenomenon, using both cell biological and reductionist biophysical perspectives. Outstanding issues in the field of mRNA condensation are disagreements of which transcripts condense in response to stress, mechanistic understanding of how mRNA condenses and accumulates into microscopically visible stress granules, and the functional consequences of mRNA condensation. Outstanding issues in the field of protein condensation are a lack of high resolution understanding of the structures of condensates, how the structures of condensates may differ in different stress contexts, and how Nature encodes condensation into a protein’s primary sequence. Furthermore, how organisms modulate condensation by altering the chemical environment of the cell remains understudied.In Chapter 2, I summarize our understanding of stress-induced condensation of mRNA and protein, detail active areas of inquiry, and raise grand challenges plaguing the field from answering these questions.
In Chapter 3, we interrogate mRNAs condensation during stress using budding yeast as a model organism. I show that most mRNAs condense following exposure to multiple divergent stresses. Rather than length being the defining predicter of mRNA condensation, we find that transcriptionally induced mRNAs escape condensation. Mechanistic work reveals that an increased abundance of ribosome-free mRNA is not sufficient to explain stress-induced mRNA condensation. Rather than simply being a byproduct of stress-triggered translational downregulation, our data supports a model in which mRNA condensation helps focus the cell’s translational machinery to produce proteins needed to mount its stress response.
In Chapter 4, I probe the molecular mechanisms of protein condensation using polyadenylate-
binding protein (Pab1 in budding yeast) as a model. I advance our understanding of Pab1 condensation mechanism by identifying putative, specific crosslinks connecting Pab1 protomers in the condensate. Supporting the thermodynamic specificity model of Pab1 condensation, I use HDX-MS to probe the hydrogen bond networks of Pab1 condensates formed at different temperatures and find that different condensation onset temperatures causes different condensate structures. HDX-MS study of Pab1 condensates from orthologs with different condensation onset temperatures informs how Nature encodes condensation in primary sequence.
In Chapter 5, I investigate how Nature may utilize transition metal signaling to modu- lation condensation. Using Pab1 from budding yeast as a model system, I find that Zn2+ specifically promotes Pab1 condensation. Transition metals may be a broadly applicable class of signaling molecules, aiding the cell to transduce stress signals into condensate formation
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HDX–MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker
Bimolecular condensation plays a role in many cellular processes. Despite considerable progress, a residue-level description of condensates has been lacking as obtaining high-resolution structural information is impeded by the condensation process itself. We overcame this issue by applying hydrogen–deuterium exchange/mass spectrometry (HDX–MS) to a canonical stress granule marker protein. We propose a sequential activation model where each domain is activated at different temperatures, executes partial unfolding, and associates only with other similarly activated domains to form the condensate, a mechanism we term thermodynamic specificity. The stress marker undergoes the same structural events upon pH- or heat-induced condensation, providing a unifying molecular portrait of stress response with the marker as a central sensor across different stresses
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An adaptive biomolecular condensation response is conserved across environmentally divergent species
Cells must sense and respond to sudden maladaptive environmental changes—stresses—to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide a remarkable view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein—a core marker of stress granules—condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry
Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae
Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M–1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed