Carrier proteins that transport 2 different types of molecules or ions may be either or antiporters.

Transporter Protein–Mediated Processes

Transporter proteins are integral membrane proteins that catalyze transfer of solutes across plasma membranes at faster rates than would occur by diffusion. Transporter proteins are a large and diverse group of molecules generally characterized by showing substrate specificity. That is, one transporter or class of transporters will predominantly transfer one substrate or class of substrates (e.g., amino acids)by having appropriate saturation kinetics (i.e., raising the concentration of a substrate solute will not infinitely increase the rate at which it is transferred on transporters)and by being competitively inhibitable (two structurally similar molecules will compete for transfer by a particular transporter protein).Transporter proteins are found most abundantly in the placenta in the microvillous and basal plasma membranes of the syncytiotrophoblast. A detailed description of all these is beyond the scope of this chapter but may be found in work by Atkinson and colleagues.57In overview, channel proteins form pores in the plasma membrane and allow diffusion of ions such as K+ and Ca2+, and transporters allow facilitated diffusion down concentration gradients, such as the GLUT1 glucose transporter (seeFig. 1.8).Exchange transporters, such as the Na+/H+ exchanger involved in pH homeostasis of the syncytiotrophoblast and fetus, andcotransporters, such as the system-A amino acid transporter—which cotransports small hydrophilic amino acids such as alanine, glycine, and serine with Na+—require the maintenance of an ion gradient through secondary input of energy, often via Na+/K+ATPase. Finally,active transporters directly utilize ATP to transfer against concentration gradients; these include the Na+/K+ATPase and the Ca2+ATPase, which pumps Ca2+ across the basal plasma membrane from syncytiotrophoblast cytosol toward the fetal circulation.

Gestational changes in the flux of solutes through transporter proteins could result from changes in the number of transporters in each plasma membrane, their turnover (i.e., the rate of binding to and release from the transporter), or their affinity for solute as well as from changes in the driving forces acting on them, such as electrochemical gradients and ATP availability. A variety of evidence shows that such developmental changes do occur. Using the technique of isolating and purifying microvillous plasma membrane and radioisotopic tracers to measure transport rates in vesicles formed from these membranes, it has been shown that the Vmax of the Na+-dependent system-A amino acid transporter increases by about fourfold per milligram of membrane protein between the first trimester and term. The activity of the system-y+ cationic amino acid (e.g., arginine, lysine) transporter increases over gestation, whereas the activity of the system-y+L transporter decreases.58 This decrease in system-y+L activity is due to a decrease in the affinity of the transporter for substrate and is accompanied by an increased expression of 4F2hc monomer of the dimer protein.58 The reason for this decline is not known but could well be associated with a specific fetal need.Glucose transporter 1 (GLUT1) expression in microvillous membrane increases between the first trimester and term.59 Na+/H+ exchanger activity is lower in first-trimester microvillous membrane vesicles compared with that at term,60 a result borne out by studies on the intrasyncytiotrophoblast pH of isolated placental villi from these two stages in gestation. Interestingly, the expression of the NHE1 isoform of this exchanger in the microvillous membrane does not change across gestation, but the expression of both of its NHE2 and NHE3 isoforms increases between weeks 14 and 18 and term.60 In contrast, no difference is apparent in Cl−/HCO3− exchanger activity or, by Western blotting, expression of its anion exchanger isoform between first trimester and term. Our understanding of how these gestational changes are regulated is currently sparse; studies in knockout mice suggest that hormones from the fetus—such as IGF-II, which signals demand for the nutrients required for growth—are important, but much further work is needed in this area.

Abstract

Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-β-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products.

Urea Transporter Proteins

Urea plays a central role in the urinary concentrating mechanism. Urea importance has been appreciated since 1934, when Gamble and colleagues initially described “an economy of water in renal function referable to urea,”355 findings which were recently confirmed and advanced in UT-A1/A3 knockout mice356 (discussed later). Many studies show that maximal urine-concentrating ability is decreased in protein-deprived or malnourished humans (and other mammals), and that urea infusion restores urine-concentrating ability (reviewed in Sands and Layton357). Urine-concentrating defects have been demonstrated in UT-A1/A3,358 UT-A2,359 UT-B,360–362 and UT-A2/UT-B knockout mice.363 Thus, an effect due to urea or UTs must be part of the mechanism by which the inner medulla concentrates urine.

Two urea transporter genes have been cloned in mammals: the UT-A (Slc14A2) gene encodes 6 protein and 9 cDNA isoforms (reviewed in Sands and Layton357), and the UT-B (Slc14A1) gene encodes 2 protein isoforms.364 The UT-A gene, which has been cloned from rodents and humans, has two promoter elements: one upstream of exon 1 and a second that is located within intron 12 and drives the transcription of UT-A2 and UT-A2b (see references365–368; also reviewed in Sands and Layton357). UT-B, which is also the Kidd blood group antigen in humans, has been cloned from humans and rodents369 (also reviewed by Sands and Layton357).

UT-A promoter I contains a tonicity enhancer (TonE) element and hyperosmolality increases its activity.366,370 UT-A1 is expressed in the terminal inner medullary collecting duct and is detected in the apical plasma membrane.367,371,372 UT-A3 is also expressed in the terminal inner medullary collecting duct; it is primarily detected in the basolateral plasma membrane but has been detected in the apical plasma membrane.373–375 UT-A2 is expressed in thin descending limbs.8,371,372,376 UT-B is expressed in descending vasa recta and red blood cells (reviewed by Sands and Layton357) (Fig. 10.16).

Vasopressin increases the phosphorylation and the apical plasma membrane accumulation of UT-A1 and of UT-A3 in rat inner medullary collecting ducts.375,377 UT-A1 is phosphorylated by vasopressin at serines 486 and 499.325,378 Both phospho-S486-UT-A1 and phospho-S499-UT-A1 are expressed predominantly in the apical plasma membrane in vasopressin-treated rat inner medullary collecting ducts.379,380 The site in UT-A3 that is phosphorylated by vasopressin has not been determined, except that neither of the two PKA consensus sites is involved.381 Vasopressin stimulates urea transport, UT-A1 phosphorylation, and apical plasma membrane accumulation through two cAMP-dependent pathways: PKA and Epac (exchange protein activated by cAMP).382 Epac increases UT-A1 phosphorylation but not at either serine 486 or 499.380

6.8 Microsomal Triacylglyceride Transfer Protein Inhibitors

MTP is critical for the formation and secretion of apoB-containing lipoproteins from the liver and intestine (Do et al., 2014) (Chapter 16). MTP transfers TAG, CE and phospholipid to apoB within the cell during the lipoprotein assembly process. Mutations in the MTP gene lead to a rare condition known as abetalipoproteinaemia, in which plasma apoB-containing lipoproteins are undetectable. In animal models, MTP inhibition results in profound reductions in plasma TAG and cholesterol concentrations (Figure 5). Although early MTP inhibitors reduced LDL cholesterol, further development of most inhibitors has been discontinued due primarily to hepatic fat accumulation. Lomitapide is the only systemic MTP inhibitor currently in development. Lomitapide substantially reduced levels of LDL cholesterol in homozygous FH and in clinical trials proved very effective in reducing LDL cholesterol in patients with homozygous FH as well as in patients with moderate hypercholesterolaemia, either as monotherapy or when combined with ezetimibe. However, variable gastrointestinal side effects, minor elevations in liver transaminase levels and increases in hepatic fat were reported. Lomitapide was recently approved for treatment of homozygous FH, as this drug was considered to provide the benefits of cardiovascular protection that outweighed risks of increased hepatic fat. An intestine-targeted MTP inhibitor has been shown to decrease both VLDL and chylomicron production and resulted in weight loss without elevations of liver enzymes or increases in hepatic fat, suggesting that this approach may be more applicable to treatment of a broader range of lipid disorders (Rached et al., 2014).

IGFBPs as Carrier Proteins

The IGFBPs complex almost all of the circulating IGF1 and IGF2 secondary to their high affinity for the IGFs (10−10 to 10−11 mol/L).285 In adults, 75% to 80% of IGFs are carried in a ternary complex consisting of one molecule of IGF plus one molecule of IGFBP3 plus one molecule of the protein ALS.286 This ternary complex is too large to leave the vascular compartment and thus extends the half-life of IGF peptides from approximately10 minutes for IGF alone to 12 to 15 hours for IGF in the ternary complex.287 Binding of IGF to IGFBP3 in a binary complex extends the half-life of IGF to 1 to 2 hours as diffusion out of the vascular compartment may occur.288

Both IGFBP3 and ALS are GH dependent, providing an additional mechanism for GH regulation of the IGF axis. GH administration to GH-deficient patients shifts IGF to the ternary complex.289 However, after IGF1 treatment alone, there is no increase in IGFBP3 levels, and ALS levels may decrease; thus IGF does not shift to the ternary complex.290 In serum of patients with GHD or GH insensitivity, little IGF is present in the 150-kDa ternary complex; the IGF present is in the IGF-IGFBP3 binary complex or is bound by other IGFBPs such as IGFBPs 1, 2, 4, or 5.291

26.2.1.1 Membrane Carrier Proteins

Membrane carrier proteins are important transmembrane polypeptide molecules which facilitate the movement of charged and polar molecules and ions across the lipid bilayer structure of the cell membranes [4]. Carrier proteins are usually found in tissues which function extensively in the absorption and excretion of molecules. Therefore, these can be found extensively in the digestive tract and the kidneys [5–7]. Carrier proteins are also important structural and functional protein molecules which play an important role in facilitated diffusion and active transport processes. These processes are two of the mechanisms introduced in the chapter on Pharmacokinetics which facilitate the distribution of drugs and other molecules to their respective drug targets (Fig. 26.1).

Transmembrane carrier proteins undergo conformation changes upon the binding of polar molecules and ions at their respective binding sites on the carrier protein which results in the facilitated movement of the molecules and ions across the cell membrane. Drugs interact with carrier proteins by occupying the binding sites of the polar molecules and ions or by affixing themselves to allosteric sites to modulate the movement of the polar molecules and ions across the cell membrane which will result in an effect [7]. Reserpine, an indole alkaloid derived from the roots of Rauwolfia serpentine, is known to block the vesicular monoamine transporter carrier protein and prevents the storage of catecholamine neurotransmitters.

Some carrier proteins can also function in the dual movement of molecules and ions across the cell membrane especially for the movement of organic molecules. These carrier proteins are categorized as symport and antiport carriers [4]. The pairing and binding of these molecules and ions are integral to the function of the carrier proteins (Fig. 26.2).

7.2.5 Transfer proteins

A nonspecific transfer protein from bovine liver has the ability to accelerate the transfer of glycosphingolipids, especially globotetraosylceramide [527]. The transfer proteins may exist in the same compartments in which the synthesis of complex oligosaccharides takes place.

Senyal and Jungalwala [528] also reported the transfer protein for galactocerebroside, sulfatide and ganglioside II3NeuAc-Gg4Cer. Metz and Radin [529] purified a cerebroside transfer protein from a cytosolic extract of bovine spleen. The molecular weight of the active protein was about 20 300, and the protein facilitated the transfer of glucosylceramide, galactosylceramide and lactosylceramide from liposomal vesicles to red cell ghosts. However, the protein did not facilitate the transfer of lecithin, cholesterol or ceramide. These data suggested this protein might stimulate the enzymes which act on cerebrosides and lactosylceramide to form sialosylgalactosylceramide, ganglioside II3NeuAc-Gg3Cer and trihexosylceramide. Abe et al. also reported a transfer protein which facilitated the transfer of galactosylceramide, lactosylceramide and glucosylceramide [530].

TLR-2 ligands built-in vaccines

Although carrier protein conjugated TACAs-based vaccines have achieved great results in preclinical trial, there are still many challenges facing the TACAs-protein glycoconjugates. First, the conjugation number of TACAs on protein is often not consistent batch-to-batch, and different conjugation number of TACAs-based vaccines resulted in vaccine efficacy variation. Moreover, carrier protein and the linker between TACAs and carrier protein are immunogenic. The strong immunogenic portion induced undesired antibodies and reduced vaccine efficacy. To overcome the problems of the carrier protein, many studies tried to conjugate TACAs to immune stimulants instead of to carrier proteins to awake immune response. Toll-like receptors (TLRs) are expressed in many different kinds of leukocytes, including dendritic cells (DC), macrophages, and B cells. They recruit protein assembly, activate downstream signaling transduction, induce cytokine production, immune cells proliferations, and other immune response. Toyokuni et al. (1994) first coupled Tn antigens to a TLR-2 ligand tripalmitoyl-glycerylcysteinylserine (Pam3Cys) as a carrier-free vaccine (Fig. 9(A)). This was the first TACAs-based vaccine that induced specific IgG antibodies to the glycan antigen without conjugation to carrier protein. Another TLR2 ligand Pam3CysSer(Lys)4 was used to couple with Tn, STn, or TF antigens on MUC1 by fragment condensation (Fig. 9(B)) (Kaiser et al., 2010). Even though this vaccine did not show a great improvement, it still induced comparable antibodies to MUC1-TT vaccine did, and the antibodies were able to recognize MUC1 glycopeptide on ELISA. Both these glycoconjugates are designed to link to Toll-like receptor 2 (TLR2) ligand, and the whole glycoconjugate binds to the TLR2 through the ligand moiety, activating TLR mediated B cells activation and resulting in B cells differentiation and IgG production.

8.12.1.1 Overview

Membrane transport proteins are involved in nutrient capture, antibiotic efflux, protein secretion, toxin production, photosynthesis, oxidative phosphorylation, photosynthesis, environmental sensing, and other vital functions in all organisms from microbe to man.1 However, membrane proteins in general are notoriously difficult to study. Owing to their hydrophobicity they are refractory to direct manipulation and can only be removed from the membrane, and their solubility maintained, in the presence of detergent.2 Such difficulties help to explain why, to date, fewer than 300 unique membrane protein structures have been resolved (see relevant examples in3,4), although the structures of over 20 000 unique soluble proteins have been solved. In fact, fewer than 1% of unique structures in the Protein Structures Database are membrane proteins, whereas they account for about 30% of all proteins in the cell.3,5 Nevertheless, there is clinical and commercial interest in inhibiting the activities of some membrane proteins, optimizing the activities of others, employing them as transducers of electrical/chemical/ mechanical energy for biosensors and in nanotechnology, etc.

TRAPP Complexes

The TRAPP complexes are similar to each other in their composition, but have different roles in vesicular trafficking events (Cai et al., 2007; Sacher et al., 2008; Hughson and Reinisch, 2010; Barrowman et al., 2010). There are three forms of the complex: TRAPPI, TRAPPII, and TRAPPIII. All TRAPP complexes have a common protein core consisting of the proteins Bet3, Bet5, Trs20, Trs23, Trs31, and Trs33. TRAPPII additionally contains the Trs65, Trs120, Trs130, and Tca17/TRAPPC2L subunits, while the TRAPPIII complex is comprised of the core proteins and Trs85. TRAPPI and TRAPPII both function in the secretory pathway in yeast; TRAPPI is required to tether ER-derived vesicles to Golgi membranes, while TRAPPII is postulated to serve as an intra-Golgi complex tether. The recently identified yeast TRAPPIII complex appears to coordinate a membrane tethering event required for autophagy (Lynch-Day et al., 2010).

TRAPPI tethers ER-derived COPII-coated vesicles to the Golgi complex through a direct interaction between the Bet3 subunit of the TRAPPI complex and the vesicle coat protein Sec23. Even though Sec23 is part of the inner layer of the COPII coat, electron microscopy images show that there are gaps in the outer Sec13/31 cage that could allow TRAPPI access to Sec23. The Bet3-Sec23 interaction appears to be essential for vesicle tethering, and this specific interaction allows for the TRAPPI complex to distinguish between COPII-coated vesicles and all other coated vesicles. In addition to their roles as tethers, the TRAPP complexes also act as Rab GEFs for the Rab GTPases Ypt1 and Ypt31/32. Structural data suggests that the central subunits of the TRAPP complexes (Bet3, Bet5, and Trs23) interact with Ypt1 and confer GEF activity. Activation of Ypt1 and Ypt31/32 by the TRAPP complexes may promote tethering and, subsequently, membrane fusion.