Thiodipeptides targeting the intestinal oligopeptide transporter as a general approach to improving oral drug delivery.

The broad substrate capacity of the intestinal oligopeptide transporter, PepT1, has made it a key target of research into drug delivery. Whilst the substrate capacity of this transporter is broad, studies have largely been limited to small peptides and peptide-like drugs. Here, we demonstrate for the first time that a diverse range of drugs can be targeted towards transport by PepT1 using a hydrolysis resistant carrier. Eleven prodrugs were synthesized by conjugating modified dipeptides containing a thioamide bond to the approved drugs ibuprofen, gabapentin, propofol, aspirin, acyclovir, nabumetone, atenolol, zanamivir, baclofen and mycophenolate. Except for the aspirin and acyclovir prodrugs, which were unstable in the assay conditions and were not further studied, the prodrugs were tested for affinity and transport by PepT1 expressed in Xenopus laevis oocytes: binding affinities ranged from approximately 0.1 to 2 mM. Compounds which showed robust transport in an oocyte trans-stimulation assay were then tested for transcellular transport in Caco-2 cell monolayers: all five tested prodrugs showed significant PepT1-mediated transcellular uptake. Finally, the ibuprofen and propofol prodrugs were tested for absorption in rats: following oral dosing the intact prodrugs and free ibuprofen were measured in the plasma. This provides proof-of-concept for the idea of targeting poorly bioavailable drugs towards PepT1 transport as a general means of improving oral permeability.

Rab11-FIP3 is a Rab11-binding protein that regulates breast cancer cell motility by modulating the actin cytoskeleton.

Cell adhesion and motility are very dynamic processes that require the temporal and spatial coordination of many cellular structures. ADP-ribosylation factor 6 (Arf6) has emerged as master regulator of endocytic membrane traffic and cytoskeletal dynamics during cell movement. Recently, a novel Arf6-binding protein known as FIP3/arfophilin/eferin has been identified. In addition to Arf6, FIP3 also interacts with Rab11, a small monomeric GTPase that regulates endocytic membrane transport. Both Arf6 and Rab11 GTPases have been implicated in regulation of cell motility. Here we test the role of FIP3 in breast carcinoma cell motility. First, we demonstrate that FIP3 is associated with recycling endosomes that are present at the leading edge of motile cells. Second, we show that FIP3 is required for the motility of MDA-MB-231 breast carcinoma cells. Third, we demonstrate that FIP3 regulates Rac1-dependent actin cytoskeleton dynamics and modulates the formation and ruffling of lamellipodia. Finally, we demonstrate that FIP3 regulates the localization of Arf6 at the plasma membrane of MDA-MB-231 cells. Based on our data we propose that FIP3 affects cell motility by regulating Arf6 localization to the plasma membrane of the leading edge, thus regulating polarized Rac1 activation and actin dynamics.

The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein recycling.

Sorting and recycling of endocytosed proteins are required for proper cellular function and growth. Internalized receptors either follow a fast constitutive recycling pathway, returning to the cell surface directly from the early endosomes, or a slow pathway that involves transport via perinuclear recycling endosomes. Slow recycling pathways are thought to play a key role in directing recycling proteins to specific locations on cell surfaces, such as the leading edges of motile cells. These pathways are regulated by various Rab GTPases, such as Rab4 and Rab11. Here we characterize the role of Rip11/FIP5, a known Rab11-binding protein, in regulating endocytic recycling. We use a combination of electron and fluorescent microscopy with siRNA-based protein knockdown to show that Rip11/FIP5 is present at the peripheral endosomes, where it regulates the sorting of internalized receptors to a slow recycling pathway. We also identify kinesin II as a Rip11/FIP5-binding protein and show that it is required for directing endocytosed proteins into the same recycling pathway. Thus, we propose that the Rip11/FIP5-kinesin-II complex has a key role in the routing of internalized receptors through the perinuclear recycling endosomes.

Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis.

The dual Rab11/Arf binding proteins, family of Rab11-interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.

The FIP3-Rab11 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis.

An integral part of cell division is the separation of daughter cells via cytokinesis. There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11- and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission.

Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins.

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.

Altered oscillatory work by ventricular myofilaments from a rabbit coronary artery ligation model of heart failure.

OBJECTIVES: Understanding the changed ability of cardiac myofilaments to produce pump work requires knowledge of kinetics of crossbridge function as well as more widely studied parameters such as Ca-sensitivity and isometric force development. We tested the hypothesis that altered crossbridge kinetics contribute to reduced myofilament work in early-stage heart failure (left ventricular dysfunction, LVD). METHODS: The sinusoidal oscillation technique can yield insights into crossbridge function. Dynamic stiffness, oscillatory work and power were assessed in chemically skinned, Ca-activated trabeculae from rabbit ventricles in early-stage failure, 8 weeks after infarction induced by coronary artery ligation (LIG). Results were compared with sham-operated controls (SH). LVD was assessed by echocardiography. RESULTS: Ca-activated force and myofilament Ca-sensitivity were not significantly altered at this early stage of LVD. In maximally Ca-activated preparations, the frequency of minimal dynamic stiffness (f(min)) was 23% lower in LIG. f(min) increases by >80% between pCa 5.8 and 4 in SH but not in LIG. Maximal phase lead and lag angles (between length and tension) were lower in LIG at frequencies near f(min), lowering oscillatory work and power. The Lissajous figures (oscillatory work loops) of imposed length vs. tension are often asymmetric near f(min). The degree of asymmetry was greater in LIG. CONCLUSIONS: Reduced capacity for mechanical power, consistent with depressed haemodynamic performance in LVD hearts, is only partially attributable to crossbridge slowing; changes in the phase relationship will also contribute. These changes are not readily attributable to known alterations in contractile protein isoforms. Some deductions are drawn about which steps in the crossbridge cycle are modified in this model of LVD. Altered cardiac myocyte Ca-transients, reported to be associated with LVD, will be translated into pump work by a contractile machinery that is functionally altered, even though isometric force and myofilament Ca-sensitivity might remain near-normal at this stage.