Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Protein phosphatase inhibitors alter cellular microtubules and reduce carbachol-dependent protein secretion in lacrimal acini.

PURPOSE: To further understand the regulation of microtubules and their function in the lacrimal gland, we investigated the effects of two serine/threonine phosphatase inhibitors, okadaic acid (300 nM-1 microM) and calyculin A (20-100 nM), on microtubules and stimulated secretion in lacrimal acini. METHODS: Primary rabbit lacrimal acini cultured for two days were utilized. Microtubule structure was probed using biochemical analysis and confocal fluorescence microscopy. Carbachol-stimulated and basal protein secretion were determined by measurement of released protein or, for pulse-chase studies, [(35)S]-protein. RESULTS: Biochemical analysis and confocal fluorescence microscopy showed that both inhibitors caused a major loss of cellular microtubules and also of acetylated (stable) microtubules. However, calyculin A was more potent than okadaic acid in causing microtubule loss. Because changes in microtubules can partially impair stimulated protein secretion in lacrimal acini, the effects of inhibitors on protein secretion were also evaluated. Both inhibitors caused a comparable dose-dependent and significant (p

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptor-mediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to alphav-integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through non-clathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrin- and non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells.

Intracellular trafficking of nonviral vectors.

Nonviral vectors continue to be attractive alternatives to viruses due to their low toxicity and immunogenicity, lack of pathogenicity, and ease of pharmacologic production. However, nonviral vectors also continue to suffer from relatively low levels of gene transfer compared to viruses, thus the drive to improve these vectors continues. Many studies on vector-cell interactions have reported that nonviral vectors bind and enter cells efficiently, but yield low gene expression, thus directing our attention to the intracellular trafficking of these vectors to understand where the obstacles occur. Here, we will review nonviral vector trafficking pathways, which will be considered here as the steps from cell binding to nuclear delivery. Studies on the intracellular trafficking of nonviral vectors has given us valuable insights into the barriers these vectors must overcome to mediate efficient gene transfer. Importantly, we will highlight the different approaches used by researchers to overcome certain trafficking barriers to gene transfer, many of which incorporate components from biological systems that have naturally evolved the capacity to overcome such obstacles. The tools used to study trafficking pathways will also be discussed.

The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway.

The interesting discovery reported here that soluble adenovirus serotype 5 (Ad5) fiber proteins enter cells without the virus was a serendipitous result during our development of Ad5 capsid proteins as nonviral gene transfer vectors. The Ad5 capsid fiber and penton proteins mediate infection. The fiber docks to a noninternalizing cell surface protein called the coxsackievirus-Ad receptor (CAR), followed by penton binding to integrins, triggering integrin-mediated endocytosis of the virus. In our previous work, we assembled the nonviral complex, 3PO, which utilized the penton to mediate gene transfer through integrin binding and endocytosis. Here, we tested whether incorporating the fiber targets 3PO to CAR, thus recapitulating the Ad5 infection pathway. As CAR is not an endocytic receptor, we were surprised to find that the fiber alone, without the penton, enabled gene transfer by binding CAR, but internalizing through an unknown mechanism. We show here that the fiber distributes to the nucleus and cytoplasm after temperature-independent uptake, whereas the penton accumulates around the nucleus after temperature-dependent uptake. Fiber uptake by HeLa cells is also actin-dependent, requires the fiber tail/shaft region, and is largely inhibited by heparin. This study raises the possibility that alternative pathways may enable both viral and nonviral cell entry.

Accumulation of catalytically active proteases in lacrimal gland acinar cell endosomes during chronic ex vivo muscarinic receptor stimulation.

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550-65) and alters the traffic of immature cathepsin B (Exp Eye Res 2004;79:665-75) in lacrimal acinar cells. To test whether active proteases aberrantly accumulate in the endosomes, cell samples were cultured 20 h with and without 10-microm carbachol (CCh), incubated with [125I]-bovine serum albumin and then lysed and analysed by subcellular fractionation. CCh decreased total cysteine protease and cathepsin S activities in the isolated lysosome, redistributing them to early endocytic and biosynthetic compartments. CCh decreased [125I] accumulation in all compartments of cells loaded in the absence of protease inhibitors; the cysteine protease inhibitor, leupeptin, prevented the endosomal decrease but not the lysosomal decrease. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography demonstrated [125I]-labelled proteolytic products in endomembrane compartments of both control and CCh-stimulated cells, even in the presence of leupeptin, but analysis indicated that CCh increased the amount in endosomes. Two-dimensional fractionation analyses suggest that the CCh-induced redistributions result from blocks in traffic to the late endosome from both the early endosome and the trans-Golgi network. Therefore, we conjecture that chronic muscarinic acetylcholine receptor stimulation leads to aberrant proteolytic processing of autoantigens in endosomes, from whence previously cryptic epitopes may be secreted to the underlying interstitial space.

Microtubule-dependent vesicle transport: modulation of channel and transporter activity in liver and kidney.

Microtubule-based vesicle transport driven by kinesin and cytoplasmic dynein motor proteins facilitates several membrane-trafficking steps including elements of endocytosis and exocytosis in many different cell types. Most early studies on the role of microtubule-dependent vesicle transport in membrane trafficking focused either on neurons or on simple cell lines. More recently, other work has considered the role of microtubule-based vesicle transport in other physiological systems, including kidney and liver. Investigation of the role of microtubule-based vesicle transport in membrane trafficking in cells of the kidney and liver suggests a major role for microtubule-based vesicle transport in the rapid and directed movement of ion channels and transporters to and from the apical plasma membranes, events essential for kidney and liver function and homeostasis. This review discusses the evidence supporting a role for microtubule-based vesicle transport and the motor proteins, kinesin and cytoplasmic dynein, in different aspects of membrane trafficking in cells of the kidney and liver, with emphasis on those functions such as maintenance of ion channel and transporter composition in apical membranes that are specialized functions of these organs. Evidence that defects in microtubule-based transport contribute to diseases of the kidney and liver is also discussed.

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase.

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.

The folate cofactor of Escherichia coli DNA photolyase acts catalytically.

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants.

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.

A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments.

To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

Increased protein phosphorylation of cytoplasmic dynein results in impaired motor function.

Inhibition of serine/threonine protein phosphatases in rat hepatocytes by okadaic acid and microcystin increased the phosphorylation of several components of the cytoplasmic dynein complex. UV light/vanadate cleavage and Western blot analysis revealed that two of these components with molecular masses of approx. 400 kDa and 74 kDa were dynein heavy- and intermediate-chains respectively. This increased phosphorylation resulted in inhibition of dynein ATPase activity, and reduced motor-dependent avidity of endosomal/lysosomal membranes for microtubules.

Identification of a novel taxol-sensitive kinase activity associated with the cytoskeleton.

The microtubule-targeted drug, taxol, enhances assembly of alphabeta tubulin dimers into microtubules. Recent work has established that taxol also elicits diverse effects on intracellular signaling. In-gel kinase assays with myelin basic protein as substrate revealed that taxol treatment significantly (P

Involvement of phosphorylation in doxorubicin-mediated myofibril degeneration. An immunofluorescence microscopy analysis.

Loss of myofilaments has been observed in both adaptive cardiac responses (i.e., hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (i.e., doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. Which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar break-down. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells.

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.

Regulation of kinesin activity by phosphorylation of kinesin-associated proteins.

The mechanochemical motor proteins of the kinesin and cytoplasmic dynein families play important roles in microtubule-based intracellular motility. Although movement and distribution of organelles like secretory granules, vesicles, endoplasmic reticulum, and chromosomes depend on the activity of these motor proteins, little is known about the regulation of this movement. We report here that the hyperphosphorylation of components of the kinesin complex by treatment with okadaic acid increases kinesin motor activity at least 2-fold. The stimulation was observed using both a granule motility assay and a microtubule gliding assay, indicating that phosphorylation enhances the activity of the motor itself, rather than the affinity of the motor for membrane organelles. Under stimulatory conditions, three proteins that co-purify with kinesin (with mobilities of 150, 79, and 73 kDa) are consistently hyperphosphorylated. Dephosphorylation of these proteins reduces kinesin activity to basal levels. Therefore, we conclude that kinesin motor activity is directly modulated by the phosphorylation state of kinesin-associated proteins.

Transferrin receptor recycling in rat hepatocytes is regulated by protein phosphatase 2A, possibly through effects on microtubule-dependent transport.

To understand the regulation of receptor-mediated endocytosis in hepatocytes, we have used two specific inhibitors of serine-threonine protein phosphatases (PP), microcystin (MCYST) and okadaic acid (OKA) as probes to alter protein phosphorylation in hepatocytes. We have then examined the impact of these changes on the specific binding and uptake of transferrin (Tf) in hepatocytes. The measurement of PP activity in hepatocyte lysates showed that OKA and MCYST shared a common inhibition of protein phosphatase 2A (PP2A). Our results showed that both OKA (250 nmol/L) and MCYST (500 nmol/L) significantly reduced Tf uptake at steady state (P < or = .05). The measurement of Tf internalization after 15 minutes in protein phosphatase inhibitor-pretreated cells revealed that the initial uptake was also significantly reduced. Binding studies showed that pretreatment with either of the phosphatase inhibitors did not result in significant changes in the K(d) for Tf binding to transferrin receptor (TfR). Additionally, no significant changes in the number of TfR in the plasma membrane were observed in phosphatase inhibitor-pretreated cells. The treatment of hepatocytes with nocodazole (NOC), which results in microtubule disassembly and inhibition of microtubule-based vesicle transport, caused comparable reductions in initial and steady state levels of transferrin accumulation. The changes in transferrin accumulation by both phosphatase inhibitors and nocodazole were accompanied by redistribution of the microtubule-anchored Golgi apparatus and lysosomal network from the perinuclear region to the cell periphery. Our data show that the regulation of Tf uptake by receptor-mediated endocytosis is mediated by PP2A and additionally may occur through regulation of microtubule-based vesicle transport.

Taxol inhibits endosomal-lysosomal membrane trafficking at two distinct steps in CV-1 cells.

Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol on endocytosis in CV-1 cells using density gradient centrifugation of membranes over sorbitol density gradients. After taxol treatment, resident endosomal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed significant (P 500 nm) vesicles found in controls. These data demonstrate that disruption of endocytic events by taxol includes the early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a subdomain of the lysosomes.