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.
INTRODUCTION
Protein and peptide therapeutics—biologics—comprise a rapidly growing sector of the modern pharmacopeia (1). Seven of the top ten highest grossing therapeutic agents in 2017 are biologics used to treat cancer (2, 3), diabetes (4), and autoimmune inflammatory disorders such as rheumatoid arthritis and Crohn’s disease (5-7). In each case, the biologic acts by stimulating, inhibiting, or replacing a protein located within plasma or on an external membrane surface (1). Not one acts within the cell cytosol or nucleus, in large part because most proteins cannot effectively breach the barrier defined by the plasma membrane (8, 9). The well-known early exceptions to this rule discovered by Löwenstein (10), Pabo (11), and Derossi (12)—the HIV trans-activator of transcription (Tat) protein and the Antennapedia homeodomain—have inspired the synthesis, study, and (in some cases) clinical evaluation (13) of literally hundreds of arginine-rich “cell-penetrating peptides” (CPPs) (14). The problem is that when added to cells, most CPPs remain trapped in endosomes and fail to achieve significant concentrations in the cytosol or nucleus (15). The inefficient delivery of proteins, peptides, and their mimetics into the mammalian cell cytosol limits their potential as therapeutics and research tools.
Recently we discovered that, when added to cells, certain small, folded miniature proteins (16, 17) derived from avian pancreatic polypeptide (aPP) or an isolated zinc-finger (ZF) domain are taken up by the endocytic pathway and released into the cytosol with unprecedented efficiencies (18, 19). The most effective molecules are defined by a discrete array of five arginine residues on a folded α-helix (20); we refer to these molecules as cell-permeant miniature proteins (CPMPs). Treatment of HeLa cells in culture with the CPMP ZF5.3 leads to a ZF5.3 concentration in the cytosol that is roughly 67% of the extracellular incubation concentration; this value is at least ten-fold higher than that achieved by HIV-Tat48-60 peptide (21) or octaarginine (Arg8) and equal to that of hydrocarbon-stapled peptides under development as protein-protein interaction inhibitors (22). Comparable improvements in cytosolic access are observed when the CPMP ZF5.3 is fused to protein cargos with significant molecular mass (23).
Here, we describe experiments that seek to understand how CPMPs like ZF5.3 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), genetic knockdowns, 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. HOPS activity is essential for cytosolic access; the closely related class C core vacuole/endosome tethering (CORVET) complex is not required. CPMPs neither inhibit nor activate HOPS activity, and we find no evidence for a direct HOPS-CPMP interaction. 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 (24). We 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 damage
The 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 CPMPs—aPP5.3 (1) and ZF5.3 (2)—were compared with those of prototypic members of three CPP families: the hydrocarbon-stapled peptide SAH-p53-8 (3) (22); D-octaarginine (D-Arg8, 4) (27), and a cyclic peptide containing both natural and unnatural amino acids, CPP12 (5) (26) (Figure 1 and Figure S1). SAH-p53-8 (3) is a hydrocarbon-stapled peptide that reaches the cell interior despite the absence of excess positive charge, D-octaarginine is the proteolytically stable enantiomer of the widely studied octapeptide L-Arg8 (27, 28), and CPP12 (5) is a cyclic peptide that reportedly reaches the cytosol with an efficiency that rivals aPP5.3 (26).
(1) (model structure from PDB: 1PPT); ZF5.3 (2) (model structure from PDB: 2EOZ); SAH-p53-8 (3) (model structure from PDB: 3V3B); D-Arg8 (4); and the cyclic peptide CPP12 (5). Unnatural amino acids: R8 = R-octenylalanine, S5 = S-pentenylalanine. See also Figure S1 and Table S1.
CPMPs and CPPs do not induce galectin recruitment at sub-micromolar concentrations
A characteristic feature of endosomes that have been damaged by invading viruses (29, 30), bacteria-triggered autophagy (31-34), or endosomolytic nanoparticles (35) is the cytosolic display of β-galactosides linked to proteoglycans that are ordinarily found on the luminal side of endolysosomal compartments (36). Once displayed to the cytosol, β-galactosides recruit cytosolic galectin (Gal) proteins (37, 38), such as Gal1, 3, 4, 8, and 9, that share a conserved β-galactoside binding site (Figure S2A) (39, 40). In particular, Gal3 and Gal8 are recruited to damaged Rab7+ and Lamp1+ endosomes that form along the degradative branch of the endocytic pathway (33, 40). Previous work has shown that endosomal damage can be detected by monitoring the translocation of eGFP fusions of Gal3 or Gal8 from the cytosol to endosome surfaces (29-34). Fusions of Gal3 or Gal8 and eGFP have also been used to visualize late endosome damage upon siRNA release from endosomes (40), screen for small molecules that induce lysosomal rupture (41), visualize endosomal leakage induced by osmocytosis and propanebetaine (iTOP) (42), and evaluate cell-permeant peptide-polymer complexes (35). However, the effects of CPPs or CPMPs on the extent of galectin-recruitment to damaged endosomes have not previously been studied.
To evaluate whether CPMPs such as aPP5.3 (1) and ZF5.3 (2) lead to the recruitment of galectins to endosomal compartments, we made use of eGFP fusions of human galectin-3 (eGFP-hGal3) and human galectin-8 (eGFP-hGal8), both of which have been used previously to detect endosomal damage (31, 39, 40). Human osteosarcoma (Saos-2) cells transiently expressing eGFP-hGal3 or eGFP-hGal8 were first treated for 1 hour with two known endosomolytic agents at concentrations reported to induce endosomal rupture and imaged using confocal microscopy to assess galectin recruitment from the cytosol to damaged endosomes (Figure S2B). These positive control reagents included Lipofectamine RNAiMAX (referred to as RNAiMAX henceforth) (16 μL/mL) and L-leucyl-L-leucine methyl ester (LLOMe) (1 mM). RNAiMAX is a cationic lipid formulation known to induce endosomal rupture to deliver siRNAs for targeted gene knockdown (40, 43). To induce visually detectable levels of endosomal rupture, we chose a concentration that is 3-fold higher than the average recommended concentration for siRNA transfection. For LLOMe, we chose a concentration, which had previously been reported to induce the desired galectin recruitment phenotype (31). Even though we routinely evaluate CPMP delivery after a 30-minute incubation period, we chose to evaluate endosomal rupture after a 60-minute incubation period to ensure detection of even low levels of rupture and galectin recruitment. The amount of endosomal recruitment was quantified using ImageJ (44) by calculating an endosomal recruitment coefficient (ERC), which was defined as the %-area of punctate fluorescence observed in a single cell divided by the total cell area, multiplied by 100 (Figure S2C). As expected (36, 40, 45), expression of eGFP-hGal3 or eGFP-hGal8 in untreated Saos-2 cells led to uniform eGFP fluorescence throughout the cytosol and nucleus with negligible punctate staining (Figure S2B). The average ERC values calculated in untreated Saos-2 cells expressing eGFP-hGal3 or eGFP-hGal8 were 2.0 ± 0.6 and 3.4 ± 0.7, respectively (Figure S2D). By contrast, treatment of Saos-2 cells with either RNAiMAX or LLOMe, both of which stimulate Gal3 and Gal8 recruitment to endolysosomal membranes in multiple cell lines (31, 33, 39, 40), led to significant punctate staining (Figure S2B). As previously reported, galectin recruitment occured within minutes and persisted for several hours (39). The average ERC values calculated when Saos-2 cells were treated with RNAiMAX were 62 ± 8 (Gal-3) and 42 ± 5 (Gal-8), values that represent increases of 30- and 14-fold over untreated cells (Figure S2B and S2D). The effects of LLOMe were even more dramatic: the average ERC values in Saos-2 cells expressing eGFP-hGal3 or eGFP-hGal8 treated with LLOMe increased 147- and 68-fold compared to untreated cells, respectively. These data confirm the utility of eGFP-hGal3 and eGFP-hGal8 for monitoring CPP or CPMP-induced endolysosomal damage in Saos-2 cells.
We next examined the effects of CPMPs aPP5.3 (1) and ZF5.3 (2) on the endosomal recruitment of eGFP-hGal3 and eGFP-hGal8. Side-by-side experiments were performed with SAH-p53-8 (3), D-Arg8 (4), and CPP12 (5). Each CPMP or CPP was tagged with a lissamine rhodamine B fluorophore (R) to enable its selective visualization alongside the eGFP-fused galectins. Galectin-expressing Saos-2 cells were treated with 600 nM 1R–5R (60 min), washed, and incubated for 30 min in CPMP/CPP-free media prior to imaging with confocal microscopy. This analysis revealed punctate red fluorescence throughout the cytosol, indicating endocytic uptake of CPPs 1R–5R, and a uniform distribution of eGFP-hGal3 and −8 throughout the cytosol and nucleus (Figure S2B). The average ERC values calculated when Saos-2 cells expressing eGFP-hGal3 or eGFP-hGal8 were treated with 600 nM CPPs 1R–5R were all less than 15 (Figure S2D), which is not significantly above the ERC of untreated Saos-2 cells. These data suggest that 1R–5R cause little or no galectin-positive endosomal damage at a treatment concentration of 600 nM.
CPMPs and CPPs do not induce endosomal leakage at concentrations below 2 μM
Although monitoring galectin recruitment reports on endolysosomal damage characterized by the cytosolic display of β-galactosides, it does not necessarily capture transient damage that can result in partial or complete endosomal leakage. To explore whether treatment of cells with CPMPs or CPPs causes endosomal leakage, we developed an assay to detect the release of lissamine rhodamine B-tagged Lys9 (Lys9R) from endosomes into the cytosol in the presence of CPPs or CPMPs. The nonapeptide Lys9 is internalized via endocytosis but not released into the cytosol under normal physiological conditions (46, 47). We verified this finding, and then used fluorescence correlation spectroscopy (FCS) (19) to quantify the concentration of Lys9R that reached the cytosol in the presence of CPMPs or CPPs (Figure S3A). Control experiments confirmed that Saos-2 cells treated with increasing concentrations of RNAiMAX (0 to 16 μL/mL) exhibited a dose-dependent increase in Lys9R, which was detected in the cytosol and nucleus using FCS. The highest concentration of RNAiMAX (16 μL/mL) yielded a 39-fold increase in the cytosolic (806 ± 87 nM) and a 46-fold increase in the nuclear (1212 ± 129 nM) concentrations of Lys9R (Figure S3B). Similar titration experiments with LLOMe yielded no detectable endosomal leakage of Lys9R, even at the highest tested LLOMe concentration (1 mM) (Figure S3C). LLOMe selectively induces lysosomal permeabilization (48, 49); it is possible that Lys9R leakage can only occur efficiently from endosomes that precede the lysosome—an observation that has also been made for the endosomal escape of siRNA lipoplexes (40) and a disulfide-bonded dimer of Tat (dfTat) (50).
Additional control experiments using FCS confirmed that the cytosolic access of each CPMP or CPP was similar in the presence and absence of unlabeled Lys9 (Lys9UL) (Figure S4A). As reported previously for experiments performed in HeLa cells, CPMPs 1R and 2R and the hydrocarbon-stapled peptide 3R reached the cytosol and nucleus with significantly greater efficiency than CPPs 4R and 5R, although cytosolic delivery was generally about 2-fold lower in Saos-2 cells than in HeLa cells (19). Miniature proteins 1R and 2R, and the hydrocarbon-stapled peptide 3R were present in the cytosol at 3- to 9-fold higher concentrations than D-Arg8 4R. We were unable to perform conclusive FCS experiments with CPPs 4R and 5R. Specifically, in cells treated with 4R and 5R, we observed diffusion times that were between 50- to 150-fold longer compared to their respective in vitro diffusion times (Figure S4B and Table S2). Diffusion times in cultured cells reportedly increase between 2- and 10-fold compared to their corresponding in vitro diffusion times (51). Increases greater than 10-fold may indicate intracellular aggregation or binding to a cellular component, preventing us from extracting meaningful parameters from the FCS data. To circumvent this problem, we performed biochemical fractionation experiments (19) to estimate the cytosolic concentrations attained by 4R and 5R.
After confirming that the presence of Lys9 had no significant effect on the ability of CPMP/CPPs 1R–5R to reach the cytosol, we evaluated the extent to which Lys9R leaked from endosomes into the cytosol or nucleus after treatment with an unlabeled CPMP or CPP (1UL–5UL) at concentrations between 0.3 and 2.4 μM. No significant leakage of Lys9R into the cytosol or nucleus was observed at CPMP or CPP concentrations below 2 μM (Figure S3D–H). At 2.4 μM, CPMPs 1UL and 2UL induced low levels of Lys9R endosomal leakage (Figure S3D and S3E), corresponding to a 6- and 10-fold increase above background for 1UL and 2UL, respectively. A similar increase in intracellular Lys9R concentration was observed at 8 μL/mL treatment with RNAiMAX (Figure S3B), a concentration that is commonly used for efficient siRNA transfection (43). The remaining polypeptides 3UL–5UL did not induce significant Lys9R leakage at any concentration tested in this assay (Figure S3F–H). Taken together, the galectin recruitment and Lys9 leakage assays demonstrate that CPMPs and CPPs 1R–5R/1UL–5UL do not induce endosomal rupture at concentrations below 2 μM. Above 2 μM, 1UL and 2UL induce low levels of galectin recruitment and Lys9R leakage, indicating that they are causing endosomal damage. Toxicity studies that monitored metabolic activity over an extended 4 h timeframe by detecting cellular ATP (CellTiter-Glo) revealed EC50 values for 1UL, 2UL, 4UL, 5UL that were >20 μM; the value for SAH-p53-8, 3UL, was 4.0 μM (Figure S4C).
Design of a genome-wide RNAi screen to probe endosomal release
The observation that sub-micromolar concentrations of CPMPs aPP5.3 (1) and ZF5.3 (2) can efficiently access the cytosol without significantly perturbing endosomal membranes raised the possibility that CPMP release from endosomes exploits a distinct cellular mechanism. To characterize this mechanism, we designed a genome-wide RNAi screen to identify candidate genes whose knockdown increase or decrease the ability of CPMP 1 to reach cytosol (Figure 2A). To quantify cytosolic delivery of 1, we made use of a previously reported glucocorticoid-induced eGFP translocation (GIGT) assay (18, 20, 52) that couples the cytosolic delivery of a molecule tagged with dexamethasone (Dex) to the nuclear translocation of a reporter protein consisting of a glucocorticoid receptor variant with exceptional affinity for dexamethasone (GR*) (53, 54) fused to eGFP (GR*-eGFP) (Figure S5A). The effect of the Dex-tagged molecule on the ratio of GFP fluorescence in the nucleus and cytosol, termed the translocation ratio (TR), can be measured with precision using an Opera High-Content Screening System and thereby provides a quantifiable readout of cytosolic access (18, 20). Previously, we demonstrated that the GIGT/Opera combination assay is associated with robust Z-factors of ≥0.5 and is therefore suitable for quantifying the cytosolic delivery of Dex-labeled CPMPs aPP5.3Dex (1Dex) and ZF5.3Dex (2Dex) in a high-throughput setting (20). Saos-2 cells stably expressing GR*-GFP (Saos-2(GIGT) cells) were prepared as described previously (20).
(A) We screened a Dharmacon SMARTpool human siRNA libraries (>18,000 genes) in Saos-2 cells stably expressing GR(C638G)-eGFP (GR*-eGFP) to identify genes whose knockdown led to significant changes in the cytosolic trafficking of CPMP 1Dex. Cytosolic localization was monitored using the GIGT assay (Holub et al., 2013) where the reporter translocation ratio (TR) refers to the ratio of the median GFP signal in the nucleus to the median signal within a 2-μm annulus of cytosol that surrounds the nucleus (Appelbaum et al., 2012). Saos-2(GIGT) cells were transfected with pools of four siRNAs (in triplicate) for 72 hours, serum-starved, and then treated with 1Dex (1 μM) for 30 minutes. The cells were fixed, stained with Hoechst 33342, and imaged on an Opera High Content Screening System. Raw TR values associated with each well were normalized to percent effect values, relative to control cells transfected with non-targeting siRNA and treated with 1Dex. We identified 428 primary hits based on the strictly standardized mean difference (β = SSMD) (Zhang et al., 2007) threshold of ±2.0. (B) Candidate genes whose knockdown inhibited the cytosolic access of 1Dex were defined by low TRs relative to control cells treated with non-targeting siRNA and 1Dex (siRNA hit type 1); candidate genes whose knockdown enhanced the cytosolic access of 1Dex were defined by high TRs relative to control cells (siRNA hit type 2); candidate genes whose knockdown led to no significant TR changes in the ability of 1Dex to reach the cytosol were excluded from further analyses (no siRNA effect). (C) An SSMD cutoff of ±2.0 resulted in an overall hit rate of 2.4%.
Before conducting the RNAi screen in a genome-wide format, we optimized cell density and siRNA transfection conditions to integrate the GIGT assay with high-content RNAi screening in Saos-2(GIGT) cells (Figure S5B). We then performed a duplicate pilot screen with 320 randomly chosen siRNAs from the Dharmacon human genome library. With this subset of siRNAs, we assessed assay performance in Saos-2(GIGT) cells treated with the CPMP 1Dex at 1 μM concentration, which yielded the highest S/B (Figure S5C). This concentration is also well below 2.4 μM, the lowest concentration at which some endosomal leakage was detected. Performance was robust across this siRNA test panel, with a mean signal-to-background (S/B) ratio of 2.3 when comparing the translocation ratios (TR) of Saos-2(GIGT) cells treated with 1Dex to untreated cells; the S/B ratio of an average siRNA screen is 2.9 (55). The observed coefficient of variation (CV), a measure of data variability, was also excellent; the 11.2% value observed was below the average observed in both small-molecule and siRNA screens (55). The calculated Z-factor (Z′) was 0.3, within the acceptable range for siRNA screens (55), and the reproducibility between replicate wells was high (Pearson correlation coefficient: r = 0.8) (Figure S5C). Collectively, these statistics are modestly better than the average of representative RNAi screens (55) and highlight the suitability of the GIGT/Opera combination assay for high-content RNAi screening.
We then used the GIGT/Opera combination assay to evaluate the effects of siRNAs targeting the majority (18,118) of human genes on the cytosolic localization of aPP5.3Dex (1Dex). Saos-2(GIGT) cells were transfected in triplicate for 72 hours with pools of four siRNAs targeting different regions of the same human mRNA, serum-starved overnight, and treated with 1 μM 1Dex for 30 minutes. The cells were fixed, stained with the nuclear dye Hoechst 33342, and the relative amounts of GR*-eGFP in the cytosol and nucleus quantified to generate a translocation ratio (TR) (Figure 2A). The raw TR of each experimental well was converted to a normalized percent effect value (Equation 10, see Supplementary Information). The average TR of Saos-2(GIGT) cells transfected with a non-targeting siRNA (RISC-Free) and treated with 1 μM 1Dex was defined as 0% effect, while that of Saos-2(GIGT) cells without 1Dex transfected with non-targeting siRNA was defined as 100% effect.
Hit identification and initial prioritization
Hits were identified from the data set by applying a strictly standardized mean difference (SSMD, β) threshold of 2.0 (56). This SSMD threshold identified 428 candidate genes that altered the GR*-eGFP translocation in the presence of 1Dex across three replicates (Figure 2A and 2C). The set of 428 candidate genes consisted of 165 genes whose knockdown inhibited and 263 genes whose knockdown enhanced GR*-eGFP translocation with 1Dex (Table S4). Genes whose knockdown inhibited GR*-eGFP translocation in the presence of 1Dex displayed average percent effect values between –26 and –100% (average = –44 ± 10%) compared to positive control cells treated with a non-targeting siRNA and 1Dex (Figure 2B: siRNA hit type 1). Genes whose knockdown increased GR*-eGFP translocation displayed average percent effect values between +50 and +250% (average = +96 ± 29%) higher than controls (Figure 2B: siRNA hit type 2). Genes whose knockdown failed to alter the GR*-eGFP translocation induced by 1Dex displayed mean percent effect values that remained unchanged when compared to control cells (Figure 2B: no siRNA hit). The primary hit rate of 2.4% (Figure 2C) falls within the range typically observed during cell-based RNAi screens (median primary hit rate of 2.3%) (57) and reflects the conservative nature of the SSMD value used for thresholding hits from the primary screen. While genes implicated in endocytosis (58) were enriched among the set of 428 initial hits (102 genes, 24% of the total), a protein-protein interaction analysis using the String database (59) revealed many interactions but no singular enriched mechanism or pathway.
To focus our attention on genes involved in the endosomal release of CPMP 1Dex, we prioritized the set of 428 initial hits to focus on genes of unknown function (29 genes) and those implicated previously in endocytic trafficking (102 genes) (Table S5). Unknown genes were identified using the publicly available GeneCards and Rat Genome Database (RGD) databases (60, 61). Genes implicated in endocytic trafficking were identified using a previously reported systems-level survey of endocytosis (58). The 297 genes lost in this filtering step encompass diverse functions. To confirm that we had successfully prioritized genes of unknown function and those implicated previously in endocytic trafficking, we cross-referenced the set of 131 prioritized genes with the Database for Annotation, Visualization and Integrated Discovery (DAVID) (62, 63). We specified the gene ontology classification “cellular compartment” to evaluate the “enrichment” of this set of 131 genes with respect to categories of subcellular organelles. The most enriched organelle categories, according to the database analysis, were the Golgi stack, the lysosomal membrane, and the late endosome. This analysis provides confidence that the set of 131 prioritized genes were worthy of subsequent study.
We recognized that some of the remaining siRNAs could be associated with the GR signaling pathway and affect the cytosolic to nuclear distribution of GR*-eGFP even in the absence of Dex or 1Dex. To identify these genes, we evaluated the individual effect of each of the four gene-specific siRNAs targeting the remaining 131 candidate genes on the distribution of GR*-GFP in non-treated Saos-2(GIGT) cells. We discarded a gene if three of the four gene-specific siRNAs significantly increased the TR compared to that measured in Saos-2(GIGT) cells transfected with a non-targeting siRNA, indicating that the gene knockdown primarily affects GR*-GFP distribution independent of the cytosolic presence of Dex or a Dex-labeled peptide. This process eliminated 61 genes from consideration (Table S6), leaving 70 genes for subsequent validation.
Next, focusing on these remaining 70 genes, we evaluated the individual effect of each of the four gene-specific siRNAs on the TR measured in Saos-2(GIGT) cells treated with either CPMP 1Dex or 2Dex, which both carry the discrete arginine array that exemplifies a penta-arg motif. We reasoned that this siRNA deconvolution procedure would identify those genes involved in cellular trafficking of both 1Dex and 2Dex, and simultaneously minimize false positives that result from siRNA off-target effects (64). Genes were retained if at least two of the four siRNAs in the pool led to significant TR changes (by one-way ANOVA test with Dunnett post hoc test) in the presence of either 1Dex or 2Dex when compared to that observed in Saos-2(GIGT) cells transfected with a non-targeting siRNA; 28 of the 70 candidate genes passed this filter (Table S7). This final set of 28 genes displayed significant enrichment for membrane compartments (19 of 28 genes) when specifying the gene ontology term “cellular compartments,” but did not exhibit enriched protein-protein interaction networks (59).
Prioritizing hits based on mechanism: genetic knockdowns that selectively mediate endosomal escape
Although the GIGT/Opera combination assay is useful for analyzing the effect on siRNA knockdowns on cytosolic access in a high-throughput mode, it is inherently qualitative and does not differentiate between knockdowns that alter CPMP/CPP uptake from those that only affect endosomal escape. To further validate and differentiate between the remaining 28 candidate genes on the basis of these criteria, we turned to two quantitative methods: flow cytometry (FC) and FCS (19). When used together, FC and FCS effectively discriminate knockdowns that alter overall CPMP/CPP uptake (quantified by FC) from those that affect endosomal escape (quantified by FCS). To evaluate the effects of each siRNA knockdown on the trafficking of CPMPs using FC and FCS, we chose to focus on CPMP 2R—while CPMPs 1R and 2R follow a similar endocytic trafficking pattern into the cell interior (18), 2R is more stable and reaches the cytosol more efficiently (19).
To evaluate the effect of the 28 candidate genes on CPMP uptake and endosomal release, Saos-2 cells were treated with pooled gene-specific siRNAs for 72 h. Transfected Saos-2 cells were then treated with 600 nM 2R for 30 minutes, lifted with trypsin to remove plasma membrane-bound peptide (18), and analyzed using FC and FCS to quantify both overall cellular uptake and the concentration of 2R that reached the cell interior (Figure 3A). Real-time quantitative polymerase chain reaction (RT-qPCR) experiments confirmed that the knockdown efficiency of each siRNA pool was >70% in Saos-2 cells (Figure S6A). Additional control experiments confirmed that neither RNAiMAX nor knockdown of the housekeeping gene GAPD affected overall CPMP uptake by Saos-2 cells (as determined by FC) or intracellular delivery of 2R (as determined by FCS) (Figure S6B).
(A) Knockdowns were achieved by transfecting Saos-2 cells with siGENOME SMARTpool siRNA and Lipofectamine RNAiMAX for 72 h according to the manufacturer’s protocol. Cells were treated with 2R (600 nM) for 30 minutes and exogenously bound peptide was removed with TrypLE Express. Cells were then evaluated using FC to assess the levels of whole-cell fluorescence intensity and using FCS to calculate the concentration of 2R in the cytosol and nucleus. (B–D) FC and FCS data illustrating the effects of gene knockdowns on both total cellular fluorescence and the cytosolic localization of 2R relative to the effects of non-targeting (RISC-Free) siRNA (NT siRNA). Knockdown of genes shown in (B) strongly inhibit (>70%) intracellular (cytosolic and nuclear) access of 2R. Knockdown of genes shown in (C) moderately inhibit (30–70%) intracellular access of 2R. Knockdown of the ARHGAP9 gene shown in (D) promotes (149%) cytosolic access of 2R. Left axis: FC = total cell uptake, fluorescence intensity per cell at 590 nm. Each data point (n) represents one biological replicate. For each FC replicate, the median fluorescence intensity at 590 nm was measured for at least 10,000 Saos-2 cells (gated for live cells). Right axis: FCS: cytosolic and nuclear concentration (nM). Each data point (n) denotes a 50-second FCS measurement recorded in a single cell. Error bars represent the standard error of the mean. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, and not significant (ns) for p > 0.05 from one-way ANOVA with Dunnett post hoc test. (E) Of the 28 final hits identified in the GIGT high-throughput screen, knockdown of six genes strongly inhibited cytosolic access, knockdown of 13 genes moderately inhibited cytosolic access, knockdown of one gene promoted cytosolic access, and knockdown of three genes had no effect on cytosolic access (false positives). The remaining five genes, which were classified as false positives, belong to a group of seven olfactory receptors (ORs), which exhibit high sequence homology (>40%) among each other. Two representative ORs were tested and displayed no significant effects compared to NT siRNA.
Knockdown of the 28 candidate genes led to significant differences in the overall uptake and intracellular delivery of 2R. Knockdown of only four of the 28 candidate genes (VPS39, SCAMP5, PIGW, and DOCK4) significantly decreased the overall uptake of 2R, as determined using FC (left axis in Figure 3B–D); none of the siRNAs significantly increased overall uptake when measured in this way (Figure 3B–D). Different trends emerged when siRNA knockdown effects were evaluated using FCS (right axis in Figure 3B–D); in this case 19 of 28 knockdowns led to a significant decrease in the concentration of 2R in the cytosol or nucleus. Knockdown of six of the 28 candidate genes strongly reduced (>70%) the delivery of 2R to the nucleus and cytosol: these genes include VPS39, SCAMP5, PXN, PIGW, MS4A4A, and LYPLA1 (Figure 3B). Knockdown of an additional 13 candidate genes moderately reduced (40–70%) the delivery of 2R to the nucleus and cytosol: these genes include TAS2R45, CASC1, KLHDC10, CSGALNACT2, INA, IL17REL, RAB2A, ZYX, INADL, ABPA3, GNG13, AVL9, and TVP23A (Figure 3C). Notably, knockdown of one candidate gene, ARHGAP9, led to a significant (+49%) increase in the delivery of 2R to the nucleus and cytosol (Figure 3D); this finding suggests that inhibitors of ARHGAP9 could improve endosomal release. It is also notable that genes whose knockdown strongly reduced the delivery of 2R encompass multiple cellular activities related to membrane homeostasis, including membrane tethering (VPS39, RAB2A), glycosylphosphatidylinositol (GPI)-anchor biosynthesis (PIGW), thioesterases (LYPLA1), and cytoskeletal genes (PXN, ZYX). The remaining eight genes consisted of DOCK4 and seven olfactory receptors (ORs: OR4C6, OR4F15, OR51E1, OR51Q1, OR52N1, OR5M8, OR8D1), which were eventually classified as false positives (Figure 3C). In summary, of the 28 genes identified in the GIGT high-throughput screen, knockdown of six strongly inhibited cytosolic access, knockdown of 13 moderately inhibited cytosolic access, and knockdown of one gene promoted cytosolic access (Figure 3E). These data provide evidence that we successfully screened and prioritized genes that primarily regulate intracellular trafficking—with 20 out 28 genes affected by FCS—and not overall uptake—with only four genes affected by FC.
Hydrocarbon-stapled α-helical peptides are a promising class of therapeutic candidates because they can exhibit improved pharmacological properties—such as increased binding affinity, proteolytic resistance, and cell permeability—compared to staple-free analogs (65-67). Although hydrocarbon-stapled peptides lack the defined array of arginine side chains that characterizes CPMPs 1 and 2, they reach the cytosol with high efficiency. In particular, the hydrocarbon-stapled peptide SAH-p53-8R (3R) achieves average intracellular concentrations (as determined by FCS in HeLa or Saos-2 cells) that are only 10–50% lower than those attained by ZF5.3R (2R), depending on the cell line used (19). To investigate whether 2 and 3 share a common mode of endosomal release, Saos-2 cells were treated with pooled siRNAs targeting the set of 20 candidate genes, treated with 3R (600 nM), and both whole-cell uptake and cytosolic delivery were quantified with FC and FCS as described above (Figure S6C).
A plot showing the percent effect of each statistically significant knockdown (that is, significant for both 2R and 3R by one-way ANOVA with Dunnett post hoc test) on the cytosolic localization of 2R or 3R showed moderate correlation (Pearson r = 0.47, P = 0.17) (Figure S7A). Knockdown of five genes—VPS39, INA, LYPLA1, KLHDC10, and CASC1 strongly (>50%) inhibited the appearance of both 2R and 3R in the cytosol (blue open circles). Knockdown of another four genes—MS4A4A, TAS2R45, RAB2A, and APBA3—resulted in a moderate (30–50%) decrease in the appearance of both 2R and 3R in the cytosol (green dots). Knockdown of AVL9, although statistically significant, did not have a strong %-effect on the ability of either 2R or 3R to reach the cytosol (Figure S7A). When the effects of these gene knockdowns on overall uptake of 2R and 3R are plotted, the effects are small, as expected (Figure S7B). Overall, the observed correlation between the effects of gene knockdowns on the cytosolic localization of CPMP 2R and hydrocarbon-stapled peptide 3R suggests that these genes play common roles in the intracellular trafficking of these two molecular families. The five genes that most strongly regulate cytosolic delivery of both 2R or 3R can be divided into four categories: (1) genes with known roles in endocytic trafficking (VPS39); (2) genes involved in the processing of post hoc translational protein modifications (LYPLA1), (3) genes involved in RNA processing (CASC1), and (4) genes with poorly characterized or unknown functions (INA and KLHDC10).
siRNA depletion of HOPS but not CORVET inhibits the cytosolic delivery of CPPs/CPMPs 1R–5R
The goal of the RNAi screen was to identify candidate genes that significantly enhance or inhibit the intracellular localization of multiple CPMPs and CPPs. One gene transcript, when depleted, strongly inhibited the cellular uptake and cytosolic delivery of both CPMP 2R and stapled peptide 3R: the VPS39 gene, also known as hVamp6. VPS39 encodes an 875-residue protein that is conserved in eukaryotes (68) and is one of six protein subunits comprising the homotypic fusion and vacuole protein sorting (HOPS) membrane tethering complex (Figure 4A). Working with VPS41 and various effector proteins (69, 70), VPS39 recruits the HOPS complex to endosomes positive for the small GTPase Rab7, such as maturing (Rab5+/Rab7+) and late (Rab7+) endosomes. The remaining four core HOPS subunits, VPS11, VPS16, VPS18, and VPS33A, serve primarily a structural role (70). A fully assembled HOPS complex is required to initiate fusion of Rab7+ maturing and late endosomes as they are converted into lysosomes (Figure 6B). Depletion of any subunit delays lysosome maturation and inhibits cargo degradation (Figure 4B) (70-72). HOPS is closely related to a structurally similar complex, the CORVET complex (Figure 4A), that precedes HOPS along the endocytic pathway by promoting the fusion of Rab5+ early and maturing endosomes (73). CORVET shares four core subunits with HOPS but contains TGFBRAP1 and VPS8 (73) in place of VPS39 and VPS41.
(A) Cartoons illustrating the protein components of the human HOPS and CORVET complexes. (B) CORVET and HOPS initiate endosomal contact and fusion along the degradative endocytic pathway. CORVET drives the fusion of early endosomes (EEs) and maturing endosomes (MEs), while HOPS drives fusion of MEs and late endosomes (LEs) to form lysosomes (LYs). (C and D) The effect of VPS39 and VPS41 knockdown (HOPS-specific subunits) compared to TGFBRAP1 and VPS8 knockdown (CORVET-specific subunits) on both overall uptake (FC) and cytosolic access (FCS) of 2R (C) and of 3R (D) relative to the effect of non-targeting siRNA (NT siRNA). Left axis: FC = total cell uptake, fluorescence intensity per cell at 590 nm. n refers to the number of biological replicates. For each FC replicate, the median fluorescence intensity at 590 nm was measured for at least 10,000 Saos-2 cells (gated for live cells). Right axis: FCS: cytosolic and nuclear concentration (nM). Each data point (n) denotes one 50-second FCS measurement recorded in the nucleus or cytosol of a single Saos-2 cell. Error bars represent the standard error of the mean. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, not significant (ns) for p > 0.05 from one-way ANOVA with Dunnett post hoc test.
Having identified VPS39 as a key regulator of the cytosolic delivery of 2R and 3R, we next asked whether the observed effect was dependent on VPS39 alone or on the other unique protein subunit of the HOPS complex: VPS41. Saos-2 cells transfected with a non-targeting (RISC-Free) or a pool of four VPS41-targeting siRNAs were treated with 2R or 3R (600 nM) for 30 minutes and analyzed using both FC and FCS as described above (Figure 4C and 4D). In our primary screen, we found that siRNAs against VPS41 displayed a mild inhibitory effect on GR*-eGFP translocation in the presence of 1Dex (27% mean percent effect, 1.58 Z-score, 1.65 SSMD), which was below our hit threshold. Here, upon VPS41 depletion, we observed a moderate decrease (–27%) in overall uptake and a strong (–71%) decrease in the average intracellular concentration for 2R (Figure 4C); these changes are comparable to those observed after VPS39 knockdown. In VPS41-depleted cells treated with hydrocarbon-stapled peptide 3R, we also observed a significant decrease in overall uptake (–27%) and in the average intracellular delivery (–55%) by FCS (Figure 4D); these changes are also similar to those observed after VPS39 knockdown. By contrast, no significant decreases in overall uptake or cytosolic localization of 2R or 3R were observed upon depletion of the CORVET-specific subunits TGFBRAP1 and VPS8 (Figure 4C and 4D); in fact, depletion of the TGFBRAP subunit led to significant increases in both overall uptake and cytosolic concentration of 2R and 3R (Figure 4C and 4D). Taken together, these data support a hypothesis in which both unique components of the HOPS complex, VPS39 and VPS41, are required to allow cytosolic access of both CPMP 2R and hydrocarbon-stapled peptide 3R. Similar trends were observed in comparable experiments performed using CPMP 1R (Figure S8A) implying that HOPS-dependent membrane tethering is also necessary for the efficient delivery of CPMP 1R to the cytosol.
Because of the unusual diffusion dynamics observed for CPPs 4R and 5R in cultured cells (Figure S4B), we were unable to perform FCS experiments to evaluate whether their delivery to the cytosol required HOPS subunits or activity. We were able to estimate their relative delivery into the cytosol on the basis of fluorescence intensity values obtained during FCS measurements (without autocorrelation); these values are proportional to the intracellular concentration in the confocal volume (Figure S8E). Knockdown of either VPS39 or VPS41 led to a significant (23–35%) decrease in whole-cell uptake of 4R or 5R (as determined by FC) and a 46–62% decrease in their average cytosolic fluorescence intensity (as determined by FCS without autocorrelation) (Figure S8B and S8C). In contrast, knockdown of the CORVET subunits TGFBRAP1 or VPS8 led to either no change (5R) or a 30% increase (4R) in overall uptake (as determined by FC) (Figure S8B and S8C). Knockdown of TGFBRAP1 led to no change in the average cytosolic fluorescence intensity of 4R or 5R (as determined by FCS without autocorrelation), while knockdown of VPS8 either stimulated (4R) or had no effect (5R) on the average cytosolic fluorescence intensity as determined by FCS without autocorrelation.
Taken together, these data show that HOPS-specific subunits VPS39 and VPS41, but not CORVET-specific subunits TGFBRAP1 and VPS8, are required for the cytosolic delivery of all CPMP/CPPs tested herein. These CPMPs and CPPs differ significantly in their intrinsic ability to reach the cytosol (Figure S4A), but in all cases this ability demands an active HOPS complex. Importantly, the more active CPMPs 1 and 2 are significantly more sensitive to knockdown of the HOPS subunit VPS39 than hydrocarbon-stapled peptide 3 or CPPs 4 and 5; knockdown of VPS39 led to 79% and 74% decrease in the cytosolic concentrations of CPMPs 1 and 2, whereas the decreases observed for hydrocarbon-stapled peptide 3 and CPPs 4 and 5 ranged from 49–56%. These data suggest that even though all peptides require HOPS-dependent fusion of late endosomes to access the cytosol, CPMPs 1 and 2 may be more strongly dependent on this pathway—or make more efficient use of it—than hydrocarbon-stapled peptides and CPPs 3–5.
HOPS remains active upon treatment with CPMPs and CPPs 1–5
Although the experiments described above indicate that efficient cytosolic delivery of CPMP/CPPs 1–5 demands the presence of a fully assembled HOPS complex, they do not discriminate between two limiting explanations for this dependence: one in which cytosolic access demands the activity of the HOPS complex and another in which cytosolic access demands the inhibition this activity. To discriminate between these two possibilities, we monitored HOPS activity in the presence of CPMPs 1UL–5UL using a previously reported fluorescence colocalization protocol that quantifies HOPS-dependent delivery of dextran to lysosomal compartments (71, 72). In this assay, the endocytic system is flooded with Alexa Fluor 488-tagged dextran for 2 h, after which the cells are washed and stained with Hoechst 33342, re-plated in dextran-free media, incubated for 1 h, stained with Magic Red, and imaged immediately by confocal microscopy (Figure S9A). Magic Red is a dye that becomes fluorescent in the presence of active cathepsin B—an enzyme exclusively localized to mature lysosomes. Dextran is only delivered to lysosomal compartments if endosomal fusion of Rab7+ endosomes occurs. This process requires a fully assembled and functional HOPS complex (71, 72).
To validate this assay, we first measured the effect of HOPS-specific subunit depletion on the colocalization of Magic Red with dextran. Saos-2 cells were transfected with siRNA pools targeting either VPS39 or VPS41, or with a non-targeting siRNA as a negative control. After 72 hours, cells were treated as described above and the colocalization of dextran and Magic Red was calculated using ImageJ (44). In untreated and non-targeting siRNA-treated Saos-2 cells, the colocalization of Alexa Fluor 488-dextran and Magic Red was characterized by Manders coefficients of 0.30 ± 0.030 and 0.23 ± 0.020, respectively; these values represent the fraction of dextran-containing vesicles that also contain cathepsin B and are comparable to those measured previously (74) (Figure S9B and S9C). As expected, depletion of HOPS-specific subunits VPS39 and VPS41 significantly reduced the colocalization of Alexa Fluor 488-dextran and Magic Red, leading to significantly lower Manders coefficients of 0.11 ± 0.024 and 0.12 ± 0.016, respectively (Figure S9B and S9C).
With a validated assay in hand, we next asked whether addition of CPMPs or CPPs 1UL–5UL had an observable effect on the intrinsic activity of the HOPS complex. Saos-2 cells were pre-treated with unlabeled CPMPs 1UL–5UL at 600 nM for 30 min and labeled with Alexa Fluor 488-dextran and Magic Red as described above (Figure S9D). In cells treated with CPMPs/CPPs 1UL–5UL, the colocalization of Alexa Fluor 488-dextran and Magic Red was characterized by Manders coefficients that ranged from 0.27 ± 0.03 (when treated with 5UL) to 0.33 ± 0.03 (when treated with 1UL), well within the range of values measured in cells that had not been treated with CPMPs or CPPs (Figure S9E and S9F). We conclude that the treatment of Saos-2 cells with CPMPs 1–5 neither inhibits nor enhances the intrinsic function of HOPS-mediated endosomal fusion. As a whole, our studies suggest that the endosomal escape of all CPMPs and CPPs tested is dependent on the presence of a functional, active HOPS complex.
How does HOPS mediate endosomal escape?
HOPS is required for the fusion of Rab7+ endosomes, a process initiated by HOPS-mediated membrane tethering and completed by the mechanical action of SNAP (soluble NSF(N-ethylmaleimide-sensitive factor) attachment protein) receptor (SNARE) proteins and the HOPS complex itself (75). Depletion of HOPS-specific subunits significantly decreases the cytosolic and nuclear concentrations of all CPMP/CPPs tested. Three limiting models are consistent with these experimental findings. The first invokes a direct interaction between a CPMP/CPP located within or on the surface of a Rab7+ late endosome and a component of the HOPS complex, perhaps VPS39 or VPS41, that effectively shuttles the CPMP or CPP from an endosome into the cytosol (model 1). The second model also positions the CPMP/CPP within or on the surface of a Rab7+ late endosome but lacks a direct HOPS-CPMP/CPP interaction; in this case HOPS facilitates escape of the CPMP/CPP during the fusion process (76, 77) (model 2). In the third model, the CPMP or CPP escapes after HOPS-dependent fusion is complete, when maturing and late endosomes have fused into compartments containing intraluminal vesicles (ILVs) (model 3). In this case, interactions between CPMPs and ILVs in the late endosome may facilitate endosomal escape; indeed, some evidence for CPP-ILV interactions have been reported (50).
Implicit in models 1 and 2 is the colocalization of a CPMP/CPP with or upon the membrane of a Rab7+ late endosome. Confocal microscopy of Saos-2 cells expressing either the early endosomal (EE) marker Rab5-GFP, the late endosomal marker (LE) Rab7-GFP, or the lysosomal (LY) marker Lamp1-GFP and treated with 2R (300 nM, 30 min) revealed low colocalization between 2R and EEs (Pearson r = 0.20 ± 0.019) but high colocalization of 2R with LEs (r = 0.55 ± 0.034) and LYs (r = 0.58 ± 0.027) (Figure 5A, 5B, 5D). However, these experiments could not resolve whether the fluorescence of 2R was localized to the endosomal lumen or the membrane. To better resolve the location of 2R, we treated Saos-2 cells with the small molecule YM201636, an inhibitor of the phosphoinositide kinase PIK-fyve that enlarges Rab7+ endolysosomes (78, 79). Again, in cells expressing GFP-labeled endosomal markers, we observed virtually no colocalization between 2R and EEs (r = 0.034 ± 0.025), good colocalization with LEs (r = 0.57 ± 0.043), and excellent colocalization with LYs (r = 0.73 ± 0.023) (Figure 5C and 5D). Line profiles across these enlarged vesicles provide no evidence for accumulation of 2R on the LE or LY membrane; instead, plots of fluorescence intensity as a function of position show clearly separable distributions of fluorescence due to GFP (green) and lissamine rhodamine B (red) (Figure 5E and 5F). These distributions suggest that 2R resides primarily within Rab7+ and Lamp1+ vesicles and not on their surface and is inconsistent with models 1 or 2 as the primary means of endosomal escape.
(A) Saos-2 cells were transduced with Rab5-, Rab7-, or Lamp1-GFP for 18 h using CellLight Reagents (BacMam 2.0). Cells were washed, incubated with CPMP 2R, stained with Hoechst 33342, lifted with TrypLE, and re-plated into microscopy slides. Cells were then incubated in media ±YM201636 for 1 hour. (B and C) Representative live-cell confocal fluorescence microscopy images of Saos-2 cells prepared as described in (A) in media without (B), or with (C) YM201636. (D) Pearson correlation coefficients characterizing colocalization of GFP markers and 2R in the presence and absence of YM201636. (E and F) Fluorescence intensity line profiles of endosomes 1–4 (see panel C). showing the relative location of emission due to 2R (red) and either (E) Rab7-GFP or (F) Lamp1-GFP. (G) Representative live-cell confocal fluorescence microscopy images of Saos-2 cells prepared as described in (A) in the presence of YM201636. White arrows identify smaller vesicles present within the boundaries of Rab7-GFP+ or Lamp1-GFP+ cells. (H) Representative live-cell confocal fluorescence image of the first frame of Movie S1. Arrows identify Lamp1-GFP+ endosomes that contain moving vesicles labeled with CPMP 2R (+YM201636), as shown in Movies S1 and S2.
Closer examination of enlarged endosomes in Rab7- or Lamp1-GFP-expressing cells revealed the presence of smaller vesicles within the boundaries of late endosomal membranes, as shown in Figure 5G and 5H and in two movies of cells expressing Lamp1-GFP and treated with 2R (white arrows in Figure 5G and 5H and Movies S1 and S2). Late endosomes are characterized, among other properties, by the presence of ILVs. To determine whether the smaller 2R-containing vesicles observed in late endosomes represent ILVs, we used a known ILV marker, the lipid N-Rh-PE, a lissamine rhodamine B-tagged version of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (80) (Figure S10A). N-Rh-PE is internalized via the endocytic pathway into ILVs (81-83) before it is eventually secreted in exosomes (80, 82, 84). As expected (80), N-Rh-PE localizes to the luminal side of Rab7-GFP+ late endosomes (r = 0.39 ± 0.050) and Lamp1-GFP+ lysosomes (r = 0.45 ± 0.031), but not to Rab5-GFP+ early endosomes (r = 0.104 ± 0.004) (Figure S10B and S10D). To image N-Rh-PE and CPMP 2 simultaneously, we tagged 2 with a silicon-rhodamine (SiR) dye that emits at 660–670 nm (85) (lissamine rhodamine B emits at 585–595 nm). Saos-2 cells treated with 2SiR alone showed a colocalization profile that mirrored that of 2R, as expected (compare Figure 5B/5D to S10C/S10E). Saos-2 cells treated with both N-Rh-PE and 2SiR showed significant colocalization (r = 0.53 ± 0.028) (Figure S10F and S10G) providing evidence that CPMP 2SiR indeed localizes to ILVs or to LEs and LYs that contain ILVs, providing support for model 3.
We also performed a four-color confocal microscopy experiment to visualize Saos-2 cells expressing either an EE, LE, or LY marker (as a GFP fusion) and treated with N-Rh-PE, 2SiR, as well as Hoechst 33342. As expected, we observed virtually no colocalization of 2SiR with EEs (r = 0.071 ± 0.014) but good colocalization with LEs (r = 0.35 ± 0.036) and LYs (r = 0.41 ± 0.037). Also as expected, we observed minimal colocalization of N-Rh-PE with EEs (r = 0.078 ± 0.023) and good colocalization with LEs (r = 0.39 ± 0.039) and LYs (r = 0.43 ± 0.045). Good colocalization between 2SiR and N-Rh-PE was observed in Saos-2 cells regardless of which endosomal marker was present (Figure S11A and S11C). To enlarge Rab7+ endolysosomes for better resolution, we again treated Saos-2 cells with YM201636 (78, 79) (Figure S11B). Pearson r values were similar in cells with and without YM201636 present (Figure S11D). By generating line profiles of endosomes displayed in Figure S11B, we again observed minimal overlap between GFP markers for Rab7 or Lamp1 and either CPMP 2SiR or ILV marker N-Rh-PE (Figure S11E and S11F). These results further support the conclusion that CPMP 2 localizes within (as opposed to on the surface of) late endosomes and lysosomes along with a marker for ILVs and provides additional support for model 3.
HOPS is required to deliver CPMP 2 to LEs and LYs
The observation that 2 colocalizes with N-Rh-PE, a lipid marker for ILVs, along with the absence of evidence that CPMP 2 localizes within the late endosomal membrane, provided circumstantial evidence for model 3, in which HOPS is required to deliver CPMPs into a cell compartment that then facilitates endosomal escape. If so, then knockdown of HOPS components (but not CORVET components) should reduce trafficking of 2SiR to LEs and LYs. To test this hypothesis, we transfected Saos-2 cells with siRNAs against HOPS subunit VPS39 and CORVET subunit TGFBRAP1. In control cells transfected with a non-targeting (RISC-Free) siRNA, we measured virtually no colocalization between 2SiR and EE marker Rab5-GFP (r = 0.05 ± 0.01), moderate colocalization with LE marker Rab7-GFP (r = 0.25 ± 0.016), and good colocalization with LY marker Lamp1-GFP (r = 0.57 ± 0.027) (Figure 6A and 6D). In HOPS-deficient cells, we also observed virtually no colocalization of 2SiR with EEs (r = 0.05 ± 0.01) and moderate colocalization with LEs (r = 0.21 ± 0.016). The colocalization with LYs (r = 0.35 ± 0.026), on the other hand, was significantly lower compared to control cells (Figure 6B and 6D). No such changes were observed in CORVET-deficient cells (virtually no colocalization of 2SiR with EEs (r = 0.083 ± 0.013); moderate colocalization with LEs (r = 0.21 ± 0.015), high colocalization with LYs (r = 0.47 ± 0.021) (Figure 6C and 6D). These data suggest that knockdown of HOPS but not CORVET inhibits trafficking of 2SiR to Lamp1+ LEs and LYs and is consistent with model 3, in which CPP/CPMPs require HOPS to reach a favorable environment for escape.
Saos-2 cells were transfected with pooled siRNAs against VPS39 or a non-targeting (NT) control siRNA (RISC-Free) for 72 h. Rab5-, Rab7-, or Lamp1-GFP were expressed by transduction with CellLight Reagents (BacMam 2.0) for 18 h. Cells were incubated with CPMP 2SiR (600 nM) for 30 min and nuclei were stained with Hoechst 33342 (300 nM) for 5 min. Cells were lifted using TrypLE Express to remove exogenously bound CPMP and re-plated for confocal microscopy. (A–C) Representative live-cell confocal microscopy images of Saos-2 cells transfected with a NT (RISC-Free) siRNA (A), VPS39-targeting siRNAs (B), or TGFBRAP1-targeting siRNAs (C). (D) Pearson correlation coefficients between GFP markers and 2SiR in non-targeting siRNA control cells, VPS39, and TGFBRAP1 knockdown cells. Scale bars = 5 μm.
DISCUSSION
Unlike many small molecules, most proteins and peptides with therapeutic potential must circumnavigate a complex journey to access the cytosol or nucleus of mammalian cells. There is compelling evidence that this journey often involves endocytosis (86, 87), the natural cellular process by which extracellular material is taken up into vesicles formed from the plasma membrane. However, the pathway into the cytosol requires not just uptake into endocytic vesicles, but also endosomal release, and this second step is widely recognized as the bottleneck hindering the efficient delivery of peptidic materials into the cytosol and nucleus (18, 88-96). We discovered several years ago that CPMPs such as ZF5.3 (18, 97, 98) overcome this bottleneck to reach the cytosol with exceptional efficiency, both with (23) and without (19) an appended protein cargo. Here, we sought to understand the cellular processes that support the trafficking of CPMPs into the cytosol. First, using two complementary cell-based assays, we ruled out the most obvious mechanism of endosomal release–a partial or complete disruption of endosomal integrity. Next, using a spectrum of tools, including a genome-wide RNA interference (RNAi) screen, quantitative fluorescence correlation spectroscopy (FCS), and live-cell, multi-color confocal imaging, we identified VPS39—a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex—as a critical determinant in the trafficking of CPMPs to the cytosol. CPMPs neither inhibit nor activate HOPS activity; indeed, HOPS activity is essential for the cytosolic access of CPMPs as well as other CPPs and hydrocarbon-stapled peptides. 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.
The activity of the HOPS complex has also been implicated in the cytosolic trafficking of numerous bacterial, fungal, and viral pathogens, including coronavirus (CoV) (99, 100), the Ebola virus (101), Fusarium graminearum (102), Candida albicans (103, 104), Cryptococcus neoformans (105), and Aspergillus nidulans (106). In particular, although their genomes lack sequences encoding a canonical penta-arg motif, both CoV and Ebola require HOPS during the later steps of their postulated mechanism of cell entry: after the viral particle has been endocytosed but before it reaches the cytosol. Passage of CoV into the cytosol requires processing of viral proteins by lysosomal proteases to enable fusion with lysosomal membranes and release into the cytosol; HOPS depletion prevents CoV from reaching the lysosome and thus fusion and release cannot occur (100). Passage of Ebola virus is regulated by HOPS in a similar manner: Ebola viral particles are unable to fuse with the lysosomal membranes in HOPS-deficient cells (101). It is important to note that, in all cases where HOPS is a required cytosolic entry factor, no direct interactions between HOPS and the pathogen have been identified. HOPS operates in a supporting role by allowing the pathogen to escape during HOPS-mediated endosomal fusion, or by providing the pathogen with access to a favorable environment for escape, fully consistent with the model proposed for the cytosolic trafficking of CPMPs and hydrocarbon-stapled peptides (model 3).
Significant experiments performed in vitro and in cultured cells provide additional support for an endosomal escape pathway that relies on the fusogenic activity of ILVs (26, 50, 78, 107). Experiments performed in vitro indicate that Tat48–60 translocates efficiently across liposomal membranes whose lipid components resemble those of late endosomal ILVs (lysobisphosphatidic acid (LBPA)/1,2-dioleoyl-sn-glycero-3-phosphocholine (PC)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 77:19:4), but not across those that resemble the late endosomal membrane (PC/PE/phosphatidylinositol (PI)/LBPA, 5:2:1:2), and not at all across those whose lipids resemble the plasma membrane (PC/PE/sphingomyelin (SM)/cholesterol, 1:1:1:1.5) (107). Similarly, a disulfide-bonded dimer of Tat (dfTat) induces leakage of the green fluorophore calcein from liposomes composed of ILV-like lipids (LBPA/PC/PE, 77:19:4), but not from liposomes that mimic EEs or the plasma membrane (PC:PE:cholesterol, 65:15:20) (50). The nuclear delivery of dfTat (as estimated by counting cells with nucleolar fluorescence staining from dfTat) is also significantly reduced in cells pre-incubated with an antibody against LBPA. LBPA is highly abundant in ILVs (108, 109) and its fusogenic activity is thought to be responsible for ILV back-fusion with endosomal membranes (110). Finally, certain cyclic CPPs, such as CPP12 (5), induce inward and outward membrane budding when added to giant unilamellar vesicles (GUVs) composed of LE lipids (PC/PE/PI/LBPA, 5:2:1:2) (26). The observed budding behavior is reminiscent of the dynamic nature of multivesicular LEs in live cells, for which ESCRT-dependent ILV inward budding and exosome outward budding are tightly connected to the unique LBPA-enriched lipid mixture observed in LEs (111). Finally, cell-permeant phosphorothioate-modified antisense oligonucleotides (PS-ASO) also colocalize with LBPA-containing ILVs inside LEs, and knockdown of ALIX protein—known to cause a reduction in cellular LBPA levels (110, 112, 113)—reduces cytosolic delivery of PS-ASO (78). Together, these data provide additional support for a model for cytosolic trafficking that requires HOPS to reach a favorable environment for endosomal escape: LEs and LYs containing ILVs enriched in the fusogenic lipid LBPA. 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.
MATERIALS AND METHODS
See Supplementary Information.
Author Contributions
A. St, J.R.L., R.F.W., and A. Sc. designed research; A. St., J.R.L., R.F.W., and S.B. performed research; A. St., J.R.L., R.F.W., and A. Sc. analyzed data and wrote the manuscript.
Acknowledgments
This work was supported by US National Institutes of Health (NIH) grants R01 GM74756 and CA 170741 (to A. Sc.). A. St. is grateful to the Howard Hughes Medical Institute for an International Student Research Fellowship. We are all grateful to Elizabeth Rhoades and Xiaohan Li for initial help with FCS, to the Yale Center for Molecular Discovery for assistance with the RNAi screen, and to Dr. Andreas Ernst for helpful comments on the manuscript.
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