A Zpr1 co-chaperone mediates folding of eukaryotic translation elongation factor 1A via a GTPase cycle.

General protein folding is mediated by chaperones that utilize ATP hydrolysis to regulate client binding and release. Zinc-finger protein 1 (Zpr1) is an essential ATP-independent chaperone dedicated to the biogenesis of eukaryotic translation elongation factor 1A (eEF1A), a highly abundant GTP-binding protein. How Zpr1-mediated folding is regulated to ensure rapid Zpr1 recycling remains an unanswered question. Here, we use yeast genetics and microscopy analysis, biochemical reconstitution, and structural modeling to reveal that folding of eEF1A by Zpr1 requires GTP hydrolysis. Furthermore, we identify the highly conserved altered inheritance of mitochondria 29 (Aim29) protein as a Zpr1 co-chaperone that recognizes eEF1A in the GTP-bound, pre-hydrolysis conformation. This interaction dampens Zpr1⋅eEF1A GTPase activity and facilitates client exit from the folding cycle. Our work reveals that a bespoke ATP-independent chaperone system has mechanistic similarity to ATPase chaperones but unexpectedly relies on client GTP hydrolysis to regulate the chaperone-client interaction.

Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis.

The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.

A TRCky TA protein delivery service snubs the UPS.

In mammals, tail-anchored (TA) proteins that are posttranslationally captured by the chaperone SGTA are triaged by the BAG6 complex into one of two fates: handoff to an ER targeting factor for membrane insertion or polyubiquitination for destruction by the proteasome. In this issue, Culver and Mariappan (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202004086) show that a fraction of newly synthesized TA proteins is polyubiquitinated in HEK293 cells independently of the BAG6 complex yet evades proteasomal degradation by undergoing deubiquitination en route to becoming stably inserted into the ER membrane.

Microwave-Generated Steam Decontamination of N95 Respirators Utilizing Universally Accessible Materials.

The SARS-CoV-2 pandemic has caused a severe, international shortage of N95 respirators, which are essential to protect healthcare providers from infection. Given the contemporary limitations of the supply chain, it is imperative to identify effective means of decontaminating, reusing, and thereby conserving N95 respirator stockpiles. To be effective, decontamination must result in sterilization of the N95 respirator without impairment of respirator filtration or user fit. Although numerous methods of N95 decontamination exist, none are universally accessible. In this work we describe a microwave-generated steam decontamination protocol for N95 respirators for use in healthcare systems of all sizes, geographies, and means. Using widely available glass containers, mesh from commercial produce bags, a rubber band, and a 1100W commercially available microwave, we constructed an effective, standardized, and reproducible means of decontaminating N95 respirators. Employing this methodology against MS2 phage, a highly conservative surrogate for SARS-CoV-2 contamination, we report an average 6-log 10 plaque forming unit (PFU) (99.9999%) and a minimum 5-log 10 PFU (99.999%) reduction after a single three-minute microwave treatment. Notably, quantified respirator fit and function were preserved, even after 20 sequential cycles of microwave steam decontamination. This method provides a valuable means of effective decontamination and reuse of N95 respirators by frontline providers facing urgent need.

Microwave-Generated Steam Decontamination of N95 Respirators Utilizing Universally Accessible Materials.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a severe, international shortage of N95 respirators, which are essential to protect health care providers from infection. Given the contemporary limitations of the supply chain, it is imperative to identify effective means of decontaminating, reusing, and thereby conserving N95 respirator stockpiles. To be effective, decontamination must result in sterilization of the N95 respirator without impairment of respirator filtration or user fit. Although numerous methods of N95 decontamination exist, none are universally accessible. In this work, we describe a microwave-generated steam decontamination protocol for N95 respirators for use in health care systems of all sizes, geographies, and means. Using widely available glass containers, mesh from commercial produce bags, a rubber band, and a 1,100-W commercially available microwave, we constructed an effective, standardized, and reproducible means of decontaminating N95 respirators. Employing this methodology against MS2 phage, a highly conservative surrogate for SARS-CoV-2 contamination, we report an average 6-log10 plaque-forming unit (PFU) (99.9999%) and a minimum 5-log10 PFU (99.999%) reduction after a single 3-min microwave treatment. Notably, quantified respirator fit and function were preserved, even after 20 sequential cycles of microwave steam decontamination. This method provides a valuable means of effective decontamination and reuse of N95 respirators by frontline providers facing urgent need.IMPORTANCE Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an increasing shortage of protective gear necessary to keep health care providers safe from infection. As of 9 April 2020, the CDC reported 9,282 cumulative cases of COVID-19 among U.S. health care workers (CDC COVID-19 Response Team, MMWR Morb Mortal Wkly Rep 69:477-481, 2020, https://doi.org/10.15585/mmwr.mm6915e6). N95 respirators are recommended by the CDC as the ideal method of protection from COVID-19. Although N95 respirators are traditionally single use, the shortages have necessitated the need for reuse. Effective methods of N95 decontamination that do not affect the fit or filtration ability of N95 respirators are essential. Numerous methods of N95 decontamination exist; however, none are universally accessible. In this study, we describe an effective, standardized, and reproducible means of decontaminating N95 respirators using widely available materials. The N95 decontamination method described in this work will provide a valuable resource for hospitals, health care centers, and outpatient practices that are experiencing increasing shortages of N95 respirators due to the COVID-19 pandemic.

Structures of human PRC2 with its cofactors AEBP2 and JARID2.

Transcriptionally repressive histone H3 lysine 27 methylation by Polycomb repressive complex 2 (PRC2) is essential for cellular differentiation and development. Here we report cryo-electron microscopy structures of human PRC2 in a basal state and two distinct active states while in complex with its cofactors JARID2 and AEBP2. Both cofactors mimic the binding of histone H3 tails. JARID2, methylated by PRC2, mimics a methylated H3 tail to stimulate PRC2 activity, whereas AEBP2 interacts with the RBAP48 subunit, mimicking an unmodified H3 tail. SUZ12 interacts with all other subunits within the assembly and thus contributes to the stability of the complex. Our analysis defines the complete architecture of a functionally relevant PRC2 and provides a structural framework to understand its regulation by cofactors, histone tails, and RNA.