HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Abstract

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7 + and Lamp1 + endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs-an environment that serves as a prerequisite for efficient endosomal escape. 4 conclude that HOPS allows CPMPs to traffic into intraluminal vesicles (ILVs), a favorable environment for endosomal escape. The identification of ILVs as a portal for passing proteins into the cytosol will aid the development of next-generation biologics that overcome the limitations imposed by cellular membranes. RESULTS Evaluating endosomal damageThe simplest way for a CPMP to escape from an endosome is if the endosome ruptures, in part or in full (25). Although there has been limited work on the effects of certain CPPs on the integrity of large unilamellar vesicles (LUVs) in vitro (26), the concentration-dependent effects of CPMPs or more traditional cell-penetrating peptides (CPPs) on endosomal integrity in cultured cells have not been thoroughly evaluated. Thus, we began our analysis with two complementary assays that together detect both subtle and severe endosomal damage in cells treated with a CPMP or CPP. One assay exploits a set of eGFP-labeled galectins to fluorescently tag damaged endosomes to enable their visualization using confocal microscopy, while the other employs a fluorescently tagged version of the nonalysine (Lys9) peptide to quantify the extent of endosome rupture in cells treated with a CPMP or CPP. In both cases, the effects of the two most efficient ...

Endosome motility defects revealed at super-resolution in live cells using HIDE probes

“…eGFP-hGal3 and -hGal8 lentiviral particles 55 were added to HeLa cells (10% FBS in DMEM, no p/s) plated on eight-well Lab-Tek chambers (Nunc, Thermo Fisher Scientific) at a density of 7 × 10 3 cells/well. After 16 hours, the cells were washed with warm DPBS three times and labeled with DiIC 16 TCO/SiR-Tz or DiIC 16’ TCO/SiR-Tz as described preciously and imaged by confocal microscopy.…”

Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT Peptide and Bis(monoacylglycero)phosphate

“…Several reports have indicated that late endosomes may serve as a site of endosomal escape. This is the case for the prototyp-ical CPP TAT, arginine-rich miniature proteins, antisense oligonucleotides, and lipoplexes (Appelbaum et al, 2012;Brock et al, 2018;Erazo-Oliveras et al, 2014;Steinauer et al, 2019;Wang et al, 2017;Yang et al, 2010). Notably, late endosomes are also a portal for the cell entry of toxins and viruses, indicating that cell delivery agents may mimic a mechanism of cell entry that biological species have evolved to exploit (Gibert et al, 2000;Helenius, 2018;Patel et al, 2016;Zaitseva et al, 2010).…”

Zinc-α2-Glycoprotein Knockout Influenced Genes Expression Profile in Adipose Tissue and Decreased the Lipid Mobilizing After Dexamethasone Treatment in Mice

“…Zinc-α2-glycoprotein (ZAG) was isolated and purified from human serum for the first time as an unknown soluble protein [1]. It has similar electrophoretic mobility to 2-globulin and contains 18 % carbohydrates [2]. Previous reports proposed that ZAG can be expressed and secreted by white adipose tissue (WAT) and brown adipose tissue (BAT), thus confirming that ZAG is a naturally secreted adipokine [3].…”