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HOPS-Dependent Endosomal Fusion Required for Efficient Cytosolic Delivery of Therapeutic Peptides and Small Proteins

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HOPS-Dependent Endosomal Fusion Required for Efficient Cytosolic Delivery of Therapeutic Peptides and Small Proteins HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins Angela Steinauera, Jonathan R. LaRochelleb, Susan L. Knoxa, Rebecca F. Wissnera, Samuel Berryc, and Alanna Schepartza,b,1 aDepartment of Chemistry, Yale University, New Haven, CT 06520-8107; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103; and cDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114 Edited by James A. Wells, University of California, San Francisco, CA, and approved November 26, 2018 (received for review July 17, 2018) Protein therapeutics represent a significant and growing compo- Recently, we discovered that, when added to cells, certain small, nent of the modern pharmacopeia, but their potential to treat folded miniature proteins (9, 10) derived from avian pancreatic human disease is limited because most proteins fail to traffic polypeptide (aPP) or an isolated zinc-finger (ZF) domain, are across biological membranes. Recently, we discovered a class of taken up into the endocytic pathway and subsequently released cell-permeant miniature proteins (CPMPs) containing a precisely into the cytosol with unprecedented efficiencies (11, 12). The most defined, penta-arginine (penta-Arg) motif that traffics readily to effective molecules are defined by a discrete array of five arginine the cytosol and nucleus of mammalian cells with efficiencies that residues on a folded α-helix (13); we refer to these molecules as rival those of hydrocarbon-stapled peptides active in animals and cell-permeant miniature proteins (CPMPs). Treatment of HeLa man. Like many cell-penetrating peptides (CPPs), CPMPs enter the cells in culture with the CPMP ZF5.3 leads to a ZF5.3 concen- endocytic pathway; the difference is that CPMPs containing a penta- tration in the cytosol that is roughly 67% of the extracellular in- Arg motif are released efficiently from endosomes, while other CPPs cubation concentration; this value is at least 10-fold higher than are not. Here, we seek to understand how CPMPs traffic from that achieved by the HIV-Tat48–60 peptide (14) or octaarginine endosomes into the cytosol and what factors contribute to the (Arg8) and equal to that of hydrocarbon-stapled peptides under efficiency of endosomal release. First, using two complementary cell- development as protein–protein interaction inhibitors (15). Im- based assays, we exclude endosomal rupture as the primary means provements in cytosolic access between two- and sixfold are ob- of endosomal escape. Next, using an RNA interference screen, served when the CPMP ZF5.3 is fused to protein cargos with fluorescence correlation spectroscopy, and confocal imaging, we VPS39— significant molecular mass (16). identify a gene encoding a subunit of the homotypic fusion Here, we describe experiments that seek to understand how and protein-sorting (HOPS) complex—as a critical determinant in the CPMPs like ZF5.3 traffic from endosomes into the cytosol, and trafficking of CPMPs and hydrocarbon-stapled peptides to the cyto- what factors contribute to the efficiency of endosomal release. sol. Although CPMPs neither inhibit nor activate HOPS function, First, using two complementary cell-based assays, we exclude HOPS activity is essential to efficiently deliver CPMPs to the cytosol. + + endosomal rupture as the primary means of endosomal escape. CPMPs localize within the lumen of Rab7 and Lamp1 endosomes and their transport requires HOPS activity. Overall, our results iden- tify Lamp1+ late endosomes and lysosomes as portals for passing Significance proteins into the cytosol and suggest that this environment is pre- requisite for endosomal escape. The potential of protein therapeutics is limited because most proteins cannot reach the cytosol. Like many cell-penetrating cell-penetrating peptides | peptidomimetics | enzyme replacement peptides (CPPs), cell-permeant miniature proteins (CPMPs) that therapy | endocytosis | protein therapeutics embody a penta-Arg motif are endocytosed; the difference is that CPMPs are released efficiently from endosomes while rotein and peptide therapeutics—biologics—comprise a other CPPs are not. Here, we report that the trafficking of rapidly growing sector of the modern pharmacopeia (1). CPMPs into the cytosol requires the homotypic fusion and P protein-sorting (HOPS) complex, a membrane-tethering com- Seven of the top 10 highest grossing therapeutic agents in + plex that fuses Rab7 endosomes. CPMPs neither inhibit nor 2017 were biologics used to treat cancer, diabetes, and autoim- activate HOPS function; instead, HOPS allows CPMPs to traffic mune inflammatory disorders, such as rheumatoid arthritis and + ’ into Lamp1 endosomes as a prerequisite for endosomal Crohn s disease. In each case, the biologic acts by stimulating, escape. The identification of Lamp1+ late endosomes and inhibiting, or replacing a protein located within plasma or on an lysosomes as portals for passing proteins into the cytosol will external membrane surface. Not one acts within the cell cytosol or aid the development of next-generation biologics that over- nucleus, in large part because most proteins cannot effectively come the limitations imposed by cellular membranes. breach the barrier defined by the plasma membrane (2). The well- known early exceptions to this rule discovered by Green and Author contributions: A. Steinauer, J.R.L., R.F.W., and A. Schepartz designed research; Loewenstein (3), Frankel and Pabo (4), and Derossi et al. (5)—the A. Steinauer, J.R.L., S.L.K., R.F.W., and S.B. performed research; A. Steinauer, J.R.L., S.L.K., R.F.W., and A. Schepartz analyzed data; and A. Steinauer, J.R.L., R.F.W., and HIV transactivator of transcription (Tat) protein and the Anten- A. Schepartz wrote the paper. — napedia homeodomain have inspired the synthesis, study, and Conflict of interest statement: A. Schepartz. and R.F.W. are named inventors of pending (in some cases) clinical evaluation (6) of hundreds of arginine-rich patent applications related to the work described. cell-penetrating peptides (CPPs) (7). The problem is that when This article is a PNAS Direct Submission. added to cells, most CPPs remain trapped in endosomes and fail Published under the PNAS license. to achieve significant concentrations in the cytosol or nucleus (8). 1To whom correspondence should be addressed. Email: [email protected]. The inefficient delivery of proteins, peptides, and their mimetics This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. into the mammalian cell cytosol limits their potential as thera- 1073/pnas.1812044116/-/DCSupplemental. peutics and research tools. Published online January 4, 2019. 512–521 | PNAS | January 8, 2019 | vol. 116 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1812044116 Downloaded by guest on September 26, 2021 Next, using an RNA interference (RNAi) screen, fluorescence These β-galactosides recruit cytosolic Gal proteins (28, 29) that correlation spectroscopy (FCS), and confocal imaging, we iden- share a conserved β-galactoside binding site (SI Appendix,Fig.S2A) tify VPS39—a gene encoding a subunit of the homotypic fusion (30, 31). In particular, Gal3 and Gal8 are recruited to damaged + + and protein-sorting (HOPS) complex—as a critical determinant Rab7 and Lamp1 endosomes that form along the degradative in the trafficking of CPMPs and hydrocarbon-stapled peptides to branch of the endocytic pathway (20, 31). Previous work has shown the cytosol. HOPS activity is essential for cytosolic access; the that endosomal damage can be detected by monitoring the trans- closely related class C core vacuole/endosome tethering (COR- location of eGFP fusions of Gal3 or Gal8 from the cytosol to VET) complex is not required. Although CPMPs neither inhibit endosome surfaces (20–26). However, the effects of CPPs or nor activate HOPS function, HOPS activity is required to effi- CPMPs on the extent of Gal-recruitment to potentially damaged ciently deliver CPMPs to the cytosol. Multicolor confocal im- endosomes have not previously been studied. + aging studies identify CPMPs within the lumen of Rab7 and To evaluate whether CPMPs, such as aPP5.3 (1) and ZF5.3 + + Lamp1 endosomes and their transport to Lamp1 endosomes (2), lead to the recruitment of Gal to endosomal compartments, requires HOPS activity. Within these compartments, CPMPs we made use of eGFP fusions of human Gal3 (eGFP-hGal3) and associate with intraluminal vesicles (ILVs). We conclude that human Gal8 (eGFP-hGal8), both of which have been used pre- HOPS is essential because it allows CPMPs to traffic into ILV- viously to detect endosomal damage (22, 30, 31). Human osteo- + + containing Rab7 and Lamp1 endosomes, where they encoun- sarcoma (Saos-2) cells transiently expressing eGFP-hGal3 or ter a favorable environment for endosomal escape. eGFP-hGal8 were first treated for 1 h with two known endo- somolytic agents at concentrations reported to induce endosomal Results rupture and then imaged using confocal microscopy to assess the Evaluating Endosomal Damage. Partial or full endosomal rupture level of Gal recruitment from the cytosol to damaged endosomes (17) is one pathway by which a CPMP could reach the cytosol or (SI Appendix,Fig.S2B). The two endosomolytic agents used as nucleus, yet the concentration-dependent effects of CPMPs or positive controls were Lipofectamine RNAiMAX (31, 32) (referred more traditional CPPs on endosomal integrity in cultured cells to as RNAiMAX
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