More Like this

1. Characterizing Soluble Protein Aggregates Using Native Mass Spectrometry Coupled with Temperature-Controlled Electrospray Ionization and Size-Exclusion Chromatography

   https://doi.org/10.1007/978-1-0716-1859-2_27  

   Muneeruddin, K.; Kaltashov, I.A.; Wang, G. (January 2022, Methods in molecular biology)

   Garcia Fruitós, E.; Arís Giralt, A. (Ed.)

   Characterization of soluble protein aggregates provides valuable information for revealing mechanisms of protein aggregation process and assessing the activity and safety of protein therapeutics. However, the noncovalent interaction, the transient nature and higher degree of structural heterogeneity of the soluble aggregation system hinders precise characterization at the molecular level. Here, we describe methods using native mass spectrometry coupled with temperature-control electrospray ionization and size-exclusion chromatography to monitor the aggregation process and profile the aggregates in detail. 

   more » « less
2. Yeast derlin Dfm1 employs a chaperone-like function to resolve misfolded membrane protein stress

   https://doi.org/10.1371/journal.pbio.3001950  

   Kandel, Rachel; Jung, Jasmine; Syau, Della; Kuo, Tiffany; Songster, Livia; Horn, Casey; Chapman, Claire; Aguayo, Analine; Duttke, Sascha; Benner, Christopher; et al (January 2023, PLOS Biology)

   Jakob, Ursula H. (Ed.)

   Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1’s previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis. 

   more » « less
3. Holistic insights from pollen omics : co-opting stress-responsive genes and ER-mediated proteostasis for male fertility

   https://doi.org/10.1093/plphys/kiab463  

   Sze, Heven; Palanivelu, Ravishankar; Harper, Jeffrey F; Johnson, Mark A (October 2021, Plant Physiology)

   Abstract Sexual reproduction in flowering plants takes place without an aqueous environment. Sperm are carried by pollen through air to reach the female gametophyte, though the molecular basis underlying the protective strategy of the male gametophyte is poorly understood. Here we compared the published transcriptomes of Arabidopsis thaliana pollen, and of heat-responsive genes, and uncovered insights into how mature pollen (MP) tolerates desiccation, while developing and germinating pollen are vulnerable to heat stress. Germinating pollen expresses molecular chaperones or “heat shock proteins” in the absence of heat stress. Furthermore, pollen tubes that grew through pistils at basal temperature showed induction of the endoplasmic reticulum (ER) stress response, which is a characteristic of stressed vegetative tissues. Recent studies show MP contains mRNA–protein (mRNP) aggregates that resemble “stress” granules triggered by heat or other stresses to protect cells. Based on these observations, we postulate that mRNP particles are formed in maturing pollen in response to developmentally programmed dehydration. Dry pollen can withstand harsh conditions as it is dispersed in air. We propose that, when pollen lands on a compatible pistil and hydrates, mRNAs stored in particles are released, aided by molecular chaperones, to become translationally active. Pollen responds to osmotic, mechanical, oxidative, and peptide cues that promote ER-mediated proteostasis and membrane trafficking for tube growth and sperm discharge. Unlike vegetative tissues, pollen depends on stress-protection strategies for its normal development and function. Thus, heat stress during reproduction likely triggers changes that interfere with the normal pollen responses, thereby compromising male fertility. This holistic perspective provides a framework to understand the basis of heat-tolerant strains in the reproduction of crops. 

   more » « less
4. Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock

   https://doi.org/10.1534/genetics.119.302237  

   Howie, Rebecca L.; Jay-Garcia, Lina Manuela; Kiktev, Denis A.; Faber, Quincy L.; Murphy, Margaret; Rees, Katherine A.; Sachwani, Numera; Chernoff, Yury O. (July 2019, Genetics)

   Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae . Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [ PSI + ], a prion isoform of the yeast translation termination factor Sup35 . This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [ PSI + ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [ PSI + ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104 -GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [ PSI + ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone. 

   more » « less
5. Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association

   https://doi.org/10.3390/ijms21145038  

   Chernova, Tatiana A.; Yang, Zhen; Karpova, Tatiana S.; Shanks, John R.; Shcherbik, Natalia; Wilkinson, Keith D.; Chernoff, Yury O. (July 2020, International Journal of Molecular Sciences)

   null (Ed.)

   Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-β ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli. 

   more » « less