EC50 beliefs are shown in Table S2. recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access, and that small protein domains containing a discrete, helical, penta-arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove PKC-IN-1 useful to explore and discover the PKC-IN-1 fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics. Introduction There is great interest in the design and discovery of synthetic molecules that influence the functions of proteins within the cytosol and nucleus of living cells.1-3 This interest is especially keen for proteins that are not enzymes, whose function depends not on covalent chemistry but rather on non-covalent interactions with other biomoleculesCnucleic acids, lipids, or other proteins. Proteins that function in this manner constitute a significant fraction of the proteome, but are notoriously difficult (albeit not impossible) to target with traditional, small molecule ligands.4-7 By contrast, proteins that function through non-covalent interactions are effectively PKC-IN-1 inhibited by peptides and small folded proteins, at least passive diffusion, at least at low concentration.27 Instead, uptake proceeds the ubiquitous and inter-dependent processes of receptor-mediated endocytosis and endosomal release.28-30 Unfortunately, most cationic peptides and proteins that engage the endocytic machinery remain trapped within vesicles where they are topologically separated from the cell interior and unable to access targets in the cytosol or nucleus.31 Intracellular function, when observed, is believed to result from the mechanistically indistinct, unpredictable, and inefficient process of endosomal escape. In accord with these early Sntb1 findings, we reported previously that small, pancreatic fold proteins containing between four and six cationic chargesC arginine side chainsCembedded within an – or PPII-helix (Figure 1) are taken up efficiently by cells into endocytic vesicles.32, 33 Endocytic uptake is favored when the arginines are clustered on an -helix within the context of a folded protein structure and is achieved without significant cytotoxicity. We reported more recently that although many pancreatic fold proteins containing four to six embedded arginines reach endocytic vesicles, very few reach the cytosol.34 Endosomal release is favored by a distinct molecular signal encoded by five dispersed but precisely arrayed arginines on an -helixCa penta-arg motif.34 The penta-arg motif is transportable into diverse protein contexts and PKC-IN-1 specifies release from vesicles characterized by the guanosine triphosphatase (GTPase) Rab5.34 Open in a separate window Figure 1 Examples of peptides and protein domains evaluated in this work. Arginine side chains are shown explicitly in those molecules drawn as ribbons.32, 33 In this work we describe two assays that were developed to help explore the structural and genetic factors that control the release of penta-arg-containing peptides, proteins, and peptide mimetics into the cytosol. In the past, identifying these factors has been constrained by the absence of rapid, robust, cell-based assays that effectively differentiate between molecules trapped within endocytic vesicles and those that escape into the cytosol.31, 36, 37 The two assays described herein are complementary. One, which we refer to as GIGI, for glucocorticoid-induced eGFP induction (Figure 2a), is an amplified assay that informs on relative cytosolic access without need for sophisticated imaging equipment or adherent cells. Because the GIGI signal is amplified by transcription and translation, this assay is especially useful when evaluating molecules whose ability to access the cytosol is low. Open in a separate window Figure 2 Overview of GIGI and GIGT assays for monitoring cytosolic localization of Dex-tagged peptides and proteins..
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- It has been well established that harboring the allele enhances dementia associated with Alzheimers disease (AD), and several studies have supported a role of proteolysis as an important factor that may contribute to this risk [2,3C10]
- [PubMed] [Google Scholar]Xiao YF, Ke Q, Wang SY, Auktor K, Yang Con, Wang GK, Morgan JP, Leaf A
- Although passively-administered hyperimmune serum conferred protection in intact birds [15,17,18], the contribution of innate defenses and cell-mediated immunity to the control of APEC in the avian host remains ill-defined
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