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Our lab loves peptides and drug discovery has recently been flooded with an interest in them. Peptides can be easy and cost efficient to make and there is an increasing number of techniques for identifying peptides that have a high affinity for challenging targets. There is, however, one major hurdle. Most peptides, linear or cyclic, struggle with cell penetrance to gain access into the cytosol or nucleus where their targets often lie. The cell membrane can be a difficult barrier to cross and even if passage into the cell is successful, peptides can wind up trapped within endosomes and unable to reach their targets. Therefore, several groups have been tackling this issue. Understanding what features and mechanisms allow for peptides to reach the cytosol is expected to unlock the full potential of these new therapeutics.

Reproduced from: Qian, Z. et al. Biochemistry. 2016. 55. 2601-2612.

A number of cell permeable miniature proteins (CPMPs) and cell penetrating peptides (CPPs) have been shown to pass through cell membranes with relatively high success. Poly-arginine motifs and cyclic structures are particularly adept at gaining entrance and peptides such as CPP-12, discovered by the Pei Group of The Ohio State University, are capable of achieving high cytosolic concentrations. Other more recent efforts have sought to quantitatively compare various peptides by a high throughput cellular assay, but an exact mechanism of cytosolic access is still unclear. Work done by the Pei Group shows CPP-12 enters the cell via endocytosis and exits endosomes through a unique physiochemical mechanism, not through non-specific lysis or leakage as previously thought. Their proposed model suggests that binding of certain peptides at the endosomal membrane induces the budding of small vesicles that pinch off into the lumen or out into the cytosol. Still, it appears that a more specific cellular mechanism should be responsible for such high deliveries into the cytosol.

With this in mind, we have been discussing the recent preprint by Schepartz Lab on bioRxiv. This work uses a rigorous RNAi screen and cellular assays to probe the mechanism by which CPMPs and CPPs can get into the cytosol. Using a pool of 5 peptides- including 2 CPMPs, Schepartz Lab’s ZF5.3 and aPP5.3 and three CPPs, Pei’s CPP-12, Aileron’s SAH-p53-8, and a common poly-arginine tag, Arg8- the group has identified the homotypic fusion and protein sorting (HOPS) complex as a main component in endosomal escape of CPMPs and CPPs. HOPS-mediated homotypic fusion is essential for cell trafficking and also required for sufficient membrane surface to generate intraluminal vesicles (ILVs). Other work has shown that ILVs located within late endosomes may provide a suitable environment for cytosolic access. This current paper suggests that while HOPS is not a direct target of these peptides, it facilitates the transfer of a CPMP into these ILVs and thereby enabling their endosomal escape. Confocal microscopy confirms that ZF5.3 localizes in ILVs and VPS39/VPS41 knockdown exhibits HOPS as essential for this activity. This early work is crucial in establishing specific molecular targets in the mechanism of endosomal escape.

There are, of course, many questions that still have to be answered before a complete mechanism of peptide endosomal escape can be elucidated. For example, if HOPS is not a direct target, is there a singular molecular entity that could be targeted to facilitate escape? Pei’s proposal in 2016 hypothesizes that CPPs access the cytosol with inward/outward budding of the endosomal membrane. Could this budding still be associated with the formation of CPP-containing ILVs? HOPs complex also appears to be essential for cytosolic access of SAH-p53-8. Does this hydrocarbon staple peptide also enter ILVs? If not, are there different mechanisms for cytosolic access available to different peptides? Also, this new evidence of ZF5.3 localizing in ILVs in late endosomes does not entirely agree with previous research that indicate ZF5.3 escapes in early endosomes. We had a hard time reconciling the two studies. That being said, there was tremendous excitement for this work in our group. Given the level sorting that occurs at in endosomes, it makes sense that specific peptide sequences might be able to take advantage of intrinsic pathway for rescuing endosomal contents: numerous bacterial, fungi, and viral pathogens do it. A total mechanism and continual research on peptide motifs that improve cell permeability/cytosol access will allow for better understanding of the future of peptide therapeutics.

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