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1.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1806200

ABSTRACT

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.


Subject(s)
Autophagy , Extracellular Vesicles , Lysosomes , Proteostasis , Autophagosomes/metabolism , Extracellular Vesicles/metabolism , Humans , Lysosomes/metabolism , SARS-CoV-2 , Sequestosome-1 Protein , Viroporin Proteins , rab27 GTP-Binding Proteins
2.
Proc Natl Acad Sci U S A ; 117(45): 28046-28055, 2020 11 10.
Article in English | MEDLINE | ID: covidwho-889324

ABSTRACT

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Drug Design , Protein Engineering/methods , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/metabolism , Binding Sites , HEK293 Cells , Humans , Molecular Docking Simulation , Mutation , Peptide Library , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Spike Glycoprotein, Coronavirus/chemistry
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