Organelle-specific Targeting Research Articles

Next step in drug delivery: getting to individual organelles

Next step in drug delivery: getting to individual organelles

Development of the Novel PEG-PE-Based Polymer for the Reversible Attachment of Specific Ligands to Liposomes: Synthesis and in Vitro Characterization

Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, whereafter successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy, and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal antibody 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound specifically with biotin-agarose. The study on the pH-dependence showed that almost 80% of the hydrazone bond was cleaved after rather brief preincubation of the immunoliposomes at pH 5 for 0.5 to 1 h. Fluorescence microscopy and flow cytometry analysis of cancer cells (HeLa and MCF-7) treated with cancer cell-specific targeting ligand mAb 2C5-bearing liposomes showed enhanced cellular binding. Studies at low pH clearly confirmed the easy cleavability of the targeting ligand from the liposomes resulting in significantly less or virtually no cellular association.

Quantitative Aspects of Intracellularly-Targeted Drug Delivery

Many pharmacological agents act intracellularly, need to be endocytosed, and reach the site of action in specific organelle to exert their action. The cell’s interior is highly compartmentalized, and complexity of the cellular endocytosis and trafficking pathways (1,2) leads to suboptimal magnitude and duration of pharmacological effects at the organelle of interest, as well as to non-specific effects due to exposure of additional organelles to the drug. Thus, attaining efficient and selective pharmacological effects for intracellularly acting drugs requires development of specialized drug delivery systems (DDS) that should be targeted to specific organelle and deliver the drug in a controlled fashion (3,4). For this purpose, particle or vesicle (liposome)-basedDDS can be used, and intracellular targeting can be achieved by decorating the drug or the DDS with organelle-specific targeting moieties. This approach relies on recognition of these moieties by the endogenous intracellular trafficking mechanisms andpreferential delivery of the drug or theDDS into specific organelle (3,4). Feasibility of targeted delivery of drugs and model compounds into individual organelles has been assessed in several studies (summarized in Table I). However, the quantitative aspects of this targeting, in terms of targeting efficiency and kinetics of drug delivery to the specific intracellular organelles, are not yet clear and should be intensively studied in order to pave the way to the clinical application of the intracellularly targeted DDSs. This commentary analyzes the existing approaches for intracellular drug targeting, their efficiency and limitations. For an introduction to intracellular drug delivery, the reader is referred to several excellent recent reviews (3,5). Delivery of DNA and RNA molecules is not in the scope of this commentary and will be mentioned only briefly.

Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.

In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

The Bax carboxy-terminal hydrophobic helix does not determine organelle-specific targeting but is essential for maintaining Bax in an inactive state and for stable mitochondrial membrane insertion

Here we address the function of the hydrophobic carboxy-terminal tail of the pro-apoptotic protein Bax. The tail is tucked into a hydrophobic pocket within the closed/inactive conformation of Bax. Apoptotic stimulation changes the Bax conformation, exposing a mitochondrial-targeting signal. We confirmed that the Bax tail alone can specifically target and anchor a passenger protein to the mitochondria. Surprisingly, we determined that the Bax tail does not play the primary targeting role in Bax mitochondrial translocation. Mutating the Bax tail to produce an ER-targeting signal had no effect on Bax mitochondrial targeting. Additionally, we demonstrated that the Bax tail has a negative regulatory effect on Bax activation. Mutations that disrupt the tail interactions with the hydrophobic pocket resulted in constitutive activation and mitochondrial targeting. Deletion of the Bax tail also resulted in an active conformation of Bax, however, mitochondrial targeting was abolished. Thus, the Bax tail is required for mitochondrial translocation. By generating a mutant-tail that cannot insert into membrane, we determined that insertion of the Bax tail is required for Bax mitochondrial targeting. Our data support a model whereby the Bax tail must be released from the pocket for activation of Bax, then functions as an anchor to stabilize Bax at the mitochondrial membrane after the initial addressing step.

Subcellular targeting: a new frontier for drug-loaded pharmaceutical nanocarriers and the concept of the magic bullet

The ability of a pharmacologically active molecule selectively to find its target is closely linked with its potential as a successful therapeutic drug. It has become increasingly evident that there are several pharmacologically active molecules that exert their action on molecular targets inside cell organelles. In the case of a drug molecule with no defined specificity for a particular organelle, the molecule would either need to have sufficiently long metabolic stability to allow for random interaction with the organelle to occur, or a targeting strategy for the intended subcellular compartment would need to be devised in order to potentiate therapeutic effect. In the case of molecules with a stronger affinity for a non-target subcellular compartment, there exists even greater need for the ability to control subcellular disposition. Subcellular or organelle-specific targeting has thus emerged as a new frontier in drug delivery. In this review selected examples of recent work are discussed that the authors believe might eventually lead to the application of pharmaceutical nanocarriers to create the next generation of ‘magic bullets’ that are capable of delivering a drug payload to a molecular target at a subcellular location.

A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan

DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient ‘petite’ cells, respectively. In wild-type cells, toxic topoisomerase I–DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1Δ cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment.

Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria.

Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways.

Determinants for Membrane Association and Permeabilization of the Coxsackievirus 2B Protein and the Identification of the Golgi Complex as the Target Organelle

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

LZ-FYVE: a novel developmental stage-specific leucine zipper, FYVE-finger protein.

We describe the molecular cloning and characterization of LZ-FYVE, a novel embryonic factor that possesses two leucine zipper motifs and a FYVE-finger domain. A partial clone of LZ-FYVE, encoding a functional leucine zipper domain, was initially isolated from a mouse embryo cDNA library by virtue of its interaction in the yeast two-hybrid system with the transcription factor ATF-2. The LZ-FYVE protein demonstrated mouse embryo-specific expression by Northern blot analysis and was detected as a nuclear protein at very restricted periods (12--14 days post-coitus) in specific tissues during embryogenesis. In particular, LZ-FYVE protein was notable in embryonic lung, cartilage, and otic capsule. Structural analysis of the deduced, full-length LZ-FYVE amino acid sequence revealed two N-terminal leucine zipper domains as well as a C-terminal FYVE-finger domain. The FYVE-finger domains specifically recognized phosphatidylinositol 3-phosphate and have been previously described only in cytoplasmic proteins involved in endosomal membrane fusion, vesicular trafficking, or organelle-specific targeting. While LZ-FYVE may have endosomal functions in the cytoplasm, because LZ-FYVE is present in the nucleus at early stages of embryonic development, it is likely that LZ-FYVE has a nuclear function.

Studying organelle physiology with fusion protein-targeted avidin and fluorescent biotin conjugates

Publisher Summary This chapter describes how the avidin chimera proteins are expressed in specific organelles of mammalian cells, the synthesis and characteristics of the Flubida dyes, and the methods used for labeling avidin-containing compartments with Flubi dyes and then studying pH regulation in mammalian organelles. Avidin chimera proteins are localized to specific compartments by appending organelle-specific targeting sequences to avidin. Cells expressing the organelle-specific avidin chimera proteins have been loaded with Flubida-2, a membrance-permeable, pH-sensitive fluorescein-biotin derivative. The targeted avidin-Flubi approach is advantageous because it allow specific targeting of a ratiometric pH-sensitive dye to the ER and Golgi of mammalian cells. The method provides flexibility in that the Flubi dyes can be modified chemically to provide different pH sensitivities that would be suitable for making measurements in both neutral organelles (e.g., ER) as well as in organelles that may vary in acidity from pH 6.4 (e.g., Golgi) to pH 4.5 (lysosomes).

4 - Protein Import Deficiencies in Human Peroxisomal Disorders

Publisher Summary This chapter provides an overview of the mechanism of protein transport into peroxisomes and presents a comparison of this knowledge to deficiencies in peroxisomal protein import in humans. In recent years, several proteins that reside in multiple subcellular compartments have been achieved by the generation of multiple protein isoforms with organelle-specific targeting information. Much interest has focused on peroxisomes as a model for the translocation of proteins across a biological membrane and on the translocation of peroxisomal proteins from the cytoplasm, where they are synthesized, into peroxisomes. The reason for the mistargeting of L-alanine:glyoxylate aminotransferase (AGT1) to mitochondria in some primary hyperoxaluria type 1 (PH1) patients is rooted in evolutionary history.