Applications of solid-state NMR to membrane proteins

Solid-state magic-angle spinning NMR of membrane proteins and protein–ligand interactions

GABAB receptors are the G-protein coupled receptors for -aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are heteromeric GPCRs which function as heterodimers composed of the GABAB1 and GABAB2 subunit. Presynaptic and postsynaptic GABAB receptors regulate Ca- and K-channels respectively via the G subunits. Furthermore, GABAB receptors can activate and inhibit adenylyl cyclase through the Gi/o and G subunits. Proteins that interact with GABAB receptors at the plasma membrane most likely contribute to the functional heterogeneity observed with native GABAB responses. We identified members of the K+ tetramerization domain containing (KCTD) protein family as novel GABAB receptor interacting proteins using immuno-affinity purification experiments from native mouse brain tissue. In separate affinity purification experiments, these proteins were also found to be associated with native Ca-channels. We have shown that native GABAB receptor exist as heteromultimers in a high molecular weight protein complex, tightly associated with KCTD8, 12, 12b, and 16. KCTDs are able to form homo- and heterodimers, thus re-enforcing their role in complex formation with the GABAB receptor. We show that the KCTD-binding site is located within the intracellular C-term of the GABAB2 subunit and that KCTDs interact with the receptor via their conserved N-termini. In addition, coassembly with KCTDs leads to alterations of GABAB receptor properties, such as an increase in agonist potency and alterations in G-protein signaling. When compared, the spatial distribution and the functional properties of KCTD8, 12, 12b, and 16 suggest overlapping but also distinct properties in the context of GABAB receptor function. In summary, this dissertation reveals KCTD8, 12, 12b, and 16 as important players within the GABAB receptor signaling complex and designates them as novel auxiliary subunits of the GABAB receptor.

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.

The chapter reviews the recent technology developments and applications of NMR in industry from 2015 to middle 2019. The chapter is divided into sections focused on applications of hyphenated NMR as a continuation of the review article published in 2016, expanding the areas of low and high field NMR for process monitoring, and including sections on specific examples for the application of solid state NMR in the pharmaceutical industry, other applications on low field NMR beyond process monitoring, contributions on NMR in the pharmaceutical, food and petroleum industry, relative configuration of molecules by NMR, and recent magnet technology development.

The aim of this thesis was to develop multidimensional high-resolution experiments that can be incorporated in a general strategy for 3D structure determination of uniformly labeled membrane proteins or fibrils (i.e. protein complexes) by MAS solid-state NMR spectroscopy. In particular, molecule orientation, dynamics and hydration have been investigated. It is proved that orientation of membrane proteins (e.g. Gramicidin A, WALP23) in aligned lipid bilayers can be obtained by recoupling N-15 CSA at fast MAS rates. A combination of scalar-coupling and dipolar-coupling based experiments (i.e. dynamics-based spectral editing) is proposed to study mobility of protein domains as shown for the cardiac modulator Phospholamban. Finally, a model of the paired helical filaments formed by protein Tau in Alzheimer`s Disease is obtained from water-protein magnetization transfer (i.e. water-edited) experiments.

Effect of Procaine HCl on ATP: Calcium-dependent Alterations in Red Cell Shape and Deformability

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal–basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking. We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1, 2 TGN, trans-Golgi network; DPP IV, dipeptidyl peptidase IV; pIgA-R, polymeric immunoglobulin A receptor; GPI, glycosyl-phosphatidylinositol; 5′NT, 5′-nucleotidase; MDCK, Madin-Darby canine kidney cells; ATP, adenosine triphosphate; Mdr1, multidrug resistance protein 1; Bsep, bile salt export pump; GFP, green fluorescent protein; Mrp2, multidrug resistant associated protein; LDL-R, low density lipoprotein receptor; AP, adaptor protein; SNARE, the soluble NSF attachment protein receptor; ERM, ezrin-radixin-moesin; TLCA, taurolithocholic acid. Three major intracellular trafficking pathways are involved in the polarized expression of plasma membrane proteins in hepatocytes. Through biogenesis, a plasma membrane protein is first synthesized in the endoplasmic reticulum, modified in Golgi, and finally sorted at the trans-Golgi network (TGN). From the TGN, they traffic along the post-Golgi biosynthetic pathway to the canalicular or sinusoidal membrane domain. After a protein reaches the plasma membrane, it can be either retained or internalized (endocytosed). After endocytosis, some proteins are transported to the opposite membrane domain of hepatocytes, a process known as transcytosis. During the past two decades, a key quest in liver cell biology has been to understand the trafficking routes and molecular mechanisms that are used by various canalicular and sinusoidal proteins as they journey along these pathways. Previous studies have demonstrated that proteins destined for the sinusoidal membrane in hepatocytes traffic from the Golgi complex to their destination directly (Fig. 1A).3 However, most canalicular proteins traffic by an indirect route, first to the sinusoidal membrane, followed by transcytosis through basolateral early endosomes and a subapical compartment, before final targeting to the canalicular domain (Fig. 1A). These canalicular proteins include single transmembrane proteins (such as dipeptidyl peptidase IV [DPP IV], aminopeptidase N, and polymeric immunoglobulin A receptor [pIgA-R]) and the glycosyl-phosphatidylinositol (GPI)-anchored proteins like 5′-nucleotidase (5′NT). Until recently, the indirect transcytotic pathway was thought to be the only route by which canalicular proteins reach their destination in hepatocytes. In contrast, most apical proteins traffic directly from TGN to the apical membrane in Madin-Darby canine kidney (MDCK) cells, a polarized kidney cell line, whereas both direct and indirect pathways operate in the retinal pigment and intestinal epithelial cells.4, 5 The post-Golgi biosynthetic trafficking and endocytic routes in hepatocytes. A hepatocyte couplet is shown with the basolateral–sinusoidal and apical–canalicular membranes separated by tight junctions (TJ). (A) Sinusoidal membrane proteins (blue circles) take a direct route (blue arrows) from the TGN to the basolateral membrane. Most canalicular proteins (green patches) are targeted initially to the basolateral membrane and then undergo transcytosis (green arrows) through the basolateral early endosome (BEE) and a subapical compartment (SAC) before reaching the canalicular membrane. (B) In contrast, Mdr1 (solid red patch) traffics directly (red arrow) from the TGN to the canalicular membrane. Bsep (hatched red patch) may traffic first to a subapical compartment (also termed as SAC) and then to the canalicular membrane. It remains unclear if Bsep and the transcytosed proteins traffic through the same SAC. (C) Some sinusoidal proteins recycle between the basolateral membrane, BEE, and SAC (dashed blue lines). The SAC functions as a common sorting station for both transcytosed and endocytosed proteins.65 Some sinusoidal proteins are targeted to late endosome (LE) and lysosomes (L) for degradation after endocytosis. At the canalicular membrane, most canalicular proteins are very slowly endocytosed with little recycling (dashed thin green lines). When an apical phosphoinositide 3 kinase is inhibited, some canalicular proteins (green patches) accumulate in lysosomes.46 (D) Mdr1 and Bsep both may recycle (dashed red lines) between the canalicular membranes and subapical compartment(s). The exact endocytic routes for Mdr1 and Bsep remain to be determined (indicated by ?). Possible intracellular compartments en route may include the apical early endosome (AEE), SAC, the apical recycling endosome (ARE), and lysosome for degradation. BC, bile canaliculi, N, nucleus. Recently, two canalicular adenosine triphosphate (ATP)–binding cassette transporters, the multidrug resistance protein 1 (Mdr1) and the bile salt export pump (Bsep), were shown to traffic directly from the Golgi to the canalicular domain (Fig. 1B). These findings were based on in vivo pulse-chase labeling in rats6 and expression of green fluorescent protein (GFP) fusion constructs in WIF-B cells, a polarized hepatic hybrid cell line.7 In addition to Mdr1 and Bsep, newly synthesized sphingolipids also reach the canalicular membrane directly from the TGN in hepatocytes.8 However, it remains unclear whether another major canalicular ATP-binding cassette transporter, the multidrug resistant-associated protein (Mrp2), which contains PDZ domains (a motif originally identified in postsynaptic protein PSD-95, Drosophila tumor suppressor Dlg and tight junction protein ZO-1; and used extensively for forming molecular scaffold as described below), also traffic directly from the Golgi to canalicular membrane. Indeed, a recent study demonstrated that Mrp2 resides in the same intracellular vesicles as pIgA-R, a canalicular protein that undergoes transcytosis.9 Much of our knowledge of the molecular mechanisms involved in polarized trafficking comes from work on epithelial cells other than hepatocytes.10 Direct basolateral targeting often involves cytoplasmic sorting signals, which can be either dileucine or tyrosine-based motifs.11 Two sinusoidal membrane proteins, the low-density lipoprotein receptor (LDL-R) and the sodium-taurocholate cotransporter, contain two tyrosine-based sorting signals.12, 13 Mutation of a critical glycine adjacent to the high-capacity tyrosine signal in LDL-R results in the mistargeting of LDL-R to the canalicular membrane in the WIF-B cells.14 Interestingly, β-turn structures, which are common to tyrosine-based basolateral sorting and endocytosis signals, are not present in the two tyrosine signals of sodium-taurocholate cotransporters.13 Basolateral sorting signals are believed to be recognized at the TGN by adaptor protein (AP) complexes, which are tetrameric protein complexes involved in both biosynthetic and endocytic sorting.15 Hepatocytes do not express μ1B, one important subunit of the AP-1B complex.16 μ1B is indispensable in the sorting of many basolateral proteins in kidney epithelial cells.17 It is not clear if hepatocytes use an unknown isoform of μ1 or a distinct AP complex to recognize the sinusoidal protein cargos. After they are bound to the AP complex, the sinusoidal proteins may be targeted and fused to the sinusoidal membrane, a process mediated in some epithelial cells by the soluble NSF (N-ethyl Maleimide-sensitive fusion protein) attachment protein receptor (SNARE) and an octomeric protein complex, which in yeast is called the exocyst-sec6/8 complex.18 The exact composition of the basolateral SNARE and exocyst complexes in hepatocytes and their roles in sinusoidal targeting have not been established. The canalicular transcytosing protein pIgA-R also takes a transcytotic route in MDCK and intestinal epithelial cells. Transcytosis of pIgA-R is stimulated by binding of immunoglobulin A and regulated by phosphorylation.19 pIgA-R contains a series of targeting signals within a cytoplasmic domain of 103 amino acids, which include a basolateral sorting signal and two tyrosine-based endocytosis signals.19 Notably, the basolateral sorting signal of pIgA-R is not dileucine or tyrosine based, and hence is different from the direct sinusoidal sorting signals discussed above. Different mechanisms other than the AP complex may determine the basolateral sorting of pIgA-R. The sorting signals in other single transmembrane proteins that undergo transcytosis are less well defined.20 Compared with direct basolateral sorting, the signals and machinery responsible for apical sorting are less well understood. Thus far, three types of apical sorting signals have been described: (1) GPI anchors, (2) transmembrane domains, and (3) N-glycans.10 In addition, the sphingolipid- and cholesterol-enriched domains, also known as lipid rafts, function as sorting platforms for apical cargos in the TGN.21 Lipid rafts are characterized by insolubility to detergent extraction at cold temperature. The enrichment of cholesterol and sphingolipid in canalicular membrane of hepatocytes has long been noted.22 Two recent independent studies demonstrate that lipid rafts mediate both transcytosis and direct apical trafficking of canalicular proteins in hepatocytes.23, 24 Slimane et al.23 showed that a GPI-anchored model protein, GPI-GFP, reaches the apical membrane of hepatic HepG2 cells by transcytosis and largely is present in lipid rafts, which are insoluble in both Triton X-100 and Lubrol WX at 4°C (Triton rafts). In contrast, two direct trafficking apical proteins, Mdr1-GFP and ATP7B (a P-type copper transporting ATPase), are contained in a distinct type of raft (Lubrol rafts), which are insoluble in Lubrol WX but are soluble in the stronger detergent, Triton X-100. Pulse-chase labeling indicated that both Triton and Lubrol rafts are formed at the TGN in HepG2 cells. Cholesterol depletion moderately increased the solubility of GPI-GFP to Triton X-100 without affecting its apical sorting. However, the same treatment led to missorting of Mdr1-GFP to the basolateral domain in HepG2 cells. Triton and Lubrol rafts also have been described in MDCK cells and segregate the GPI-anchored proteins from multispanning transmembrane proteins in the apical membrane.25 Nyasae et al.24 examined the solubility of a number of WIF-B cell apical proteins to Triton X-100 at 4°C. Two GPI-anchored proteins, 5′NT and CD59, are virtually insoluble to Triton X-100. In contrast, the single transmembrane proteins DDP IV and pIgA-R are completely soluble. Thus, not all of these canalicular proteins are contained in rafts, although they all use the indirect transcytotic pathway. Other mechanisms in addition to rafts must determine their indirect apical sorting. Moreover, the detergent-insoluble apical proteins seem to enter lipid rafts at different stages in the transcytotic pathway and with different kinetics.24 In this study, cholesterol depletion after 60 minutes severely impaired transcytosis of both raft-associated (CD59) and nonassociated proteins (DDP IV), presumably by blocking transcytotic flux from basolateral endosomes.24 Interestingly, cholesterol depletion did not affect the apical trafficking of GPI-GFP after 180 minutes in HepG2 cells in the study by Slimane et al.23 It is conceivable that the model protein GPI-GFP may not reflect faithfully the apical sorting of endogenous GPI-anchored canalicular proteins like CD59 and 5′NT. Despite this discrepancy, these two studies23, 24 together show that lipid rafts mediate the apical trafficking of some canalicular proteins in hepatocytes and that cholesterol is probably required for both Triton and Lubrol rafts. Furthermore, these two studies emphasize the complexity of membrane microdomains in the plasma membrane of hepatocytes. Judged by the sole criteria of detergent insolubility, the canalicular membrane consists of at least two types of rafts and a pool of single transmembrane proteins, some of which may be partly raft associated.23, 24 Lipid rafts also exist in the sinusoidal membrane of hepatocytes because some transcytosing proteins may have been incorporated into rafts before their transit to the sinusoidal membrane. In addition, caveolae, a special type of raft, have been detected at microvilli beneath the sinusoidal membranes of hepatocytes26 and contain raft-resident signaling proteins such as Ras and protein kinase C.27 Taken together, these studies suggest that both the canalicular and sinusoidal membranes can be viewed as a mosaic of raft-associated and nonassociated microdomains. A comprehensive analysis of detergent solubility of all canalicular and sinusoidal proteins will be required to appreciate fully the diversity of microdomains in the plasma membrane of hepatocytes and their functional implication. It also would be interesting to examine directly rafts and other microdomains in live hepatic cells using high-resolution fluorescence microscopy, which has been used successfully to examine raft-resident proteins in several cell types.28 Although it is not yet certain how raft mechanisms mediate the apical sorting of canalicular proteins, the work by Slimane et al.23 suggests that Lubrol rafts may segregate multispanning transmembrane proteins and may influence their direct apical sorting in HepG2 cells. It remains to be determined if other ATP-binding cassette proteins that traffic directly to the canalicular domain, such as Bsep, reside in the same type of lipid rafts. As discussed above, most GPI-anchored and single transmembrane proteins traffic directly from the TGN to the apical membrane in MDCK cells but indirectly via transcytosis to the canalicular membrane in hepatocytes. Why is there such a difference? Presumably, the plasma membrane and sorting mechanisms are organized differently between these two cell types. For example, secretory forms of DPP IV and pIgA-R (lacking the transmembrane regions) are secreted across the apical membrane in MDCK cells,29, 30 but across the basolateral domain in hepatic WIF-B cells.31 In hepatocytes, most proteins are secreted primarily across the sinusoidal domain. Canalicular proteins that undergo transcytosis seem to be sorted similarly at the TGN as the sinusoidal secreted proteins, that is, they are initially targeted to the sinusoidal domain. Basolateral endosomes also have been proposed to function as a sorting station for proteins that undergo transcytosis to the apical membrane in hepatocytes.31 In contrast, Mdr1 and Bsep must be sorted differently because they traffic directly to the canalicular domain. How these proteins exactly are sorted through the TGN remains to be determined. Transcytosis is not a phenomenon unique to hepatocytes because a number of apical proteins are delivered partly by transcytosis and partly by direct targeting to the apical membrane in intestinal epithelial cells.32 Transcytosis also seems to be regulated during epithelial differentiation. In Fisher rat thyroid cells, transcytosis functions as a major apical sorting mechanism for DPP IV in early epithelial differentiation, but is replaced by direct apical targeting in more differentiated epithelial cells.33 It is worth noting that few mammalian cells have robust protein secretion across the basolateral membrane like hepatocytes. Therefore, sinusoidal protein secretion, which is established early in hepatocyte differentiation and persists in adult liver, may be the reason why transcytosis is a major apical sorting mechanism for most canalicular proteins in adult hepatocytes. Interestingly, Bsep, a direct trafficking canalicular protein, is detected in rat liver only after birth,34 consistent with the notion that direct apical targeting may be established in hepatocytes at a later stage of differentiation. In contrast, Mrp2 is detected at the bile canaliculi before birth,34 consistent with the possibility that Mrp2 may be sorted via the transcytotic rather than the direct pathway. To understand fully the mechanistic difference in polarized trafficking between hepatocytes and other epithelial cells, a comprehensive comparison of the molecules and complexes involved in protein sorting will be required. For example, soluble NSF (N-ethyl maleimide-sensitive fusion protein) attachment protein-23, a key component of the SNARE complex, is present at both the apical and basolateral membranes of many epithelial cells but is absent from the canalicular membrane of hepatocytes.3 The apical SNARE complex mediates the docking and fusion of Triton rafts (containing GPI-anchored proteins) to the apical membrane in MDCK cells.35 It remains to be determined if the absence of soluble NSF attachment protein-23 in the canalicular membrane contributes to the preference of transcytosis as a major apical delivery mechanism in hepatocytes. Several sinusoidal membrane proteins, such as LDL-R, the transferrin receptor, and the asialoglycoprotein receptor, have a dynamic residence at the plasma membrane and undergo endocytosis (Fig. 1C).36 What about canalicular proteins? Tuma et al.37 recently demonstrated that several indirect pathway proteins (DPP IV, aminopeptidase N, and 5′NT) as well as MRP2 largely are retained at the apical membrane in differentiated hepatic WIF-B cells (Fig. 1C). However, these proteins recycle between plasma membrane and a novel intracellular compartment in less differentiated Fao and Clone 9 cells, which contain only would-be apical proteins. These data suggest that apical retention mechanisms are established during hepatocyte differentiation. In contrast, Mdr1-GFP and Bsep-GFP (which presumably reflect Mdr1 and Bsep) undergo dynamic endocytosis and recycle between the canalicular membrane and a subapical region when expressed in WIF-B cells (Fig. 1D).7, 38 Why is there such a difference in the dynamics of plasma membrane proteins in hepatocytes? First, the actin-based cytoskeleton is thought to be responsible for retention of domain-specific proteins.39, 40 Proteins like ezrin-radixin-moesin (ERM) bind to both actin and single transmembrane proteins directly41 and interact with multispanning proteins like Mrp2 via their PDZ domains.42 Therefore, ERM proteins mediate the formation of a molecular scaffold between apical membrane and actin cytoskeleton, which leads to the retention of these canalicular proteins. The degree of retention for single transmembrane proteins and Mrp2 may be different because disruption of actin filaments affected their localization differently in WIF-B cells.37 In addition, knockout of radixin in mice led to a significant decrease in the canalicular membrane expression of Mrp2 but not DDP IV, Mdr1, or Bsep.43 At present, it is not known how ERM or other retention mechanisms operate underneath the sinusoidal membrane. Second, dynamic membrane proteins like the transferrin receptor and raft-resident proteins are endocytosed by the clathrin- and caveolae-mediated pathways, respectively.44, 45 At present, it is not known which pathways mediate the fast apical endocytosis of Mdr1 and Bsep and the slow apical endocytosis of indirect pathway proteins and Mrp2. Several mechanisms may explain why Mdr1 and Bsep recycle rather than being retained in the canalicular membrane like Mrp2: (1) Mdr1 and possibly Bsep reside in distinct lipid rafts,23 which may confer different lateral diffusion properties, and (2) Mdr1 and Bsep do not posses PDZ motifs and presumably do not bind to tethering ERM proteins. Despite a tight apical retention mechanism, slow endocytosis and subsequent targeting to lysosomes may still function as a means to remove and degrade the indirect pathway proteins like aminopeptidase N (2%–3% degraded per hour37) and Mrp2 from the canalicular membrane. Inhibition of Vps34p, an apical phosphoinositide 3 kinase by wortmannin or injection of inhibitory Vps34p antibodies, led to accumulation of apical proteins like 5′-NT and MRP2 in lysosomal vacuoles in WIF-B cells.46, 47 Phosphoinositide 3 kinase generates secondary lipid messengers from inositol, which in turn regulates many steps of both biosynthetic and endocytic sorting. Thus far, we have considered how hepatic membrane polarity is maintained. How is such a complex polarity generated in hepatocytes during liver development and in particular how are the transcytotic delivery and apical retention mechanisms established? A number of electron microscopic studies have documented the ultrastructural changes in hepatocyte progenitor cells and hepatocytes at different stages of liver development.48-50 In chick embryos, the earliest liver parenchymal cells are not polarized.48 As the liver bud invades the perihepatic mesoderm, the parenchymal cells become polarized at day 5 when canaliculi-specific antigens are first detected. By day 7, the liver rudiment shows ultrastructural characteristics of bile canaliculi, and a canalicular network can be seen.48 From this point on, hepatocytes are polarized, but the bile canaliculi do not attain a fully several after Interestingly, in hepatocytes, intracellular bile canaliculi have been that may canalicular domains and may be to the intracellular apical compartment in Fao cells described by Tuma et al.37 The development of membrane polarity is to the process of hepatocyte differentiation. To understand fully the molecular involved in the of hepatocyte a analysis of the trafficking cytoskeleton, and signaling molecules at different stages of hepatic differentiation are required. on recent work from other epithelial we that the of hepatic polarity involves the in In hepatocyte progenitor cells, would-be sinusoidal and canalicular membrane proteins are sorted at the TGN into distinct vesicles by basolateral sorting signals and raft mechanisms (Fig. cells the these proteins may be into the but these microdomains are with other in the plasma membrane. Some intracellular compartments may be formed and may contain the would-be canalicular proteins, an stage before formation of the canalicular membrane. As the liver bud invades the perihepatic mesoderm, the of hepatocytes is by or (Fig. or the a process mediated by cell molecules like may be to the of and may to the of domain-specific or both a membrane or a that in turn may a number of (Fig. As by et and these include the establishment of apical junction complexes junction and the of signaling and polarized attachment of actin and which then to the of the cytoskeleton in the would-be canalicular and sinusoidal proteins distinct on the plasma membrane, followed by the of apical and basolateral the canalicular and basolateral domains are formed in hepatocytes (Fig. and actin cytoskeleton also are established in a polarized shown in A hypothetical model for the of membrane polarity during hepatocyte differentiation. Several stages of during hepatocyte differentiation. (A) hepatocyte progenitor cells are not polarized. The would-be sinusoidal (blue circles) and would-be canalicular proteins (green and red for indirect and direct pathway proteins, are sorted at the TGN and are in the plasma membrane. Some intracellular apical compartments may be formed that contain canalicular proteins. (B) As the liver bud invades the perihepatic mesoderm, of hepatocytes is by exocyst complexes may be to the to the subsequent of domain-specific (C) a membrane which the formation of apical The actin network to the complexes, to the and of the cytoskeleton in hepatocytes. canalicular and sinusoidal proteins traffic to the plasma membrane (indicated by arrows) and the canalicular and sinusoidal (D) membrane polarity is fully established in hepatocytes. The and of are The subapical actin cytoskeleton network to tight junctions and the canalicular membrane, forming as a the canalicular domain. BC, bile tight During this specific that liver differentiation may influence the expression and distribution of specific membrane targeting molecules soluble NSF attachment retention molecules the ERM and the of cytoskeleton the subapical actin these the mechanisms for polarized protein For example, hepatocyte and the formation of tight junctions during and may function similarly in liver differentiation. of to of Fao cells these cells to bile canaliculi and to functional of cell Thus bile which are secreted only at the later stage of liver development, also may affect hepatocyte membrane polarity and the of bile canaliculi, possibly by signaling through To these at the molecular will to in of the expression and signaling between the and Fao at different during development of cell polarity may interesting about when and how transcytosis and direct apical trafficking is established in to hepatocyte of hepatocyte polarity is a of liver by or injection of leads to a of canalicular proteins such as pIgA-R and DPP IV to the domain and accumulation in subapical (Fig. and treatment also the of transcytotic with an in both the number and of transcytotic vesicles within the subapical intracellular in which may from fusion of transcytotic vesicles and may bile canaliculi both and These intracellular seem to the apical compartments in less differentiated hepatic cells and liver, as discussed above. In liver, the tight junctions are also the between bile and Taken together, these suggest that transcytosis of canalicular proteins and leads to a or of membrane The mechanisms responsible for altered cell polarity in are understood. However, the of to the trafficking of Bsep to the canalicular domain is to be in hepatocytes in and can both transcytosis and the expression of Mrp2 in the canalicular The these inhibitory of and the of and to the canalicular membrane, which may in turn affect the trafficking of Mrp2. It is conceivable that bile also may directly or indirectly affect the trafficking of other plasma membrane proteins in hepatocytes. Much work is in membrane polarity and polarized trafficking in hepatocytes. leads to the of canalicular proteins (green patches) to the sinusoidal and lateral domains, of transcytotic vesicles red and the of intracellular junctions also are a between the and taurolithocholic the targeting and of transcytosed proteins and Mrp2 to the canalicular membrane, with the of to the canalicular membrane. the inhibitory of and of to the canalicular membrane. studies described in this have significant into how sinusoidal and canalicular proteins traffic to and reside in the plasma membrane. However, the molecular mechanisms by which hepatocytes and maintain membrane polarity still remain understood. more of the polarized sorting machinery is required. by the of hepatocyte differentiation, we may into how hepatocyte polarity is generated and how the preference of hepatocytes to proteins to the apical canalicular membrane by transcytosis is established. studies of polarized trafficking in hepatocytes have been largely because of developments such as described in this the a understanding of this complex yet vital of hepatocytes to and maintain their polarity in and The and Tuma for during the of this and and for We to are not because of of

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

Background:Therapeutic monoclonal antibodies used in Chronic lymphocytic leukemia (CLL) act through complement‐mediated cytotoxicity and other mechanisms, and thus depend on the complement (C) availability and activity. Recently we showed abnormal Western analysis pattern of the C5 component in some CLL patients, that was associated with high basal levels of C activation markers, and decreased activity of the classical pathway (CP). We hypothesized that the CP may be constantly activated, and thus exhausted, in a sub‐group of CLL patients, identified by the abnormal appearance of C5.Aims:The aim was to study the circulating abnormal C5 and its association with the observed decrease in CP activity, and to search for potential CP‐activating factors in CLL plasma.Methods:Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). Activities of the CP and of the CP C5‐convertase were studied. C5, C5a and C5b were studied by Western blot analysis. The presence of high molecular weight (HMW) protein complexes, such as IgM or hexameric‐IgG, as potential CP activators was assessed by sera fractionation (gel‐filtration chromatography) followed by C activation with the obtained HMW, and low molecular weight (LMW) fractions. The levels of IgM were measured. Data from patients with abnormal C5 was compared to normal NC subjects and to patients with normal C5.Results:In patients with abnormal C5, activities of the CP and of the CP C5‐convertase were decreased, compared to NC subjects and to patients with normal C5. The abnormal C5 was identified as a complex of immunoglobulin with C5a (Ig‐C5a). In agreement, the high levels of C activation markers showed negative and significant correlation with activity of the CP, but not with the alternative pathway. The data on C activation by the HMW fraction indicated the presence of IgG‐aggregates, and not IgM, as the C activating factor in sera.Summary/Conclusion:In some CLL patients, factors such as IgG‐aggregates that are present in sera constantly trigger the activation of the CP. This constant activation results in the release of activation products, such as C5a, that form the complex Ig‐C5a. Another result is the exhaustion of the CP and the inability to exert CP activity that is comparable to normal subjects. The data provides a potential prognostic tool that may personalize therapy by identifying a sub‐group of CLL patients who display Ig‐C5a and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

We describe the partial purification of an endonuclease from calf thymus that nicks phage PM2 DNA irradiated with UV doses producing only a few pyrimidine dimers per molecule. It has much less activity on DNA that has been subjected to enzymatic photoreactivation after UV irradiation. The calf thymus endonuclease is different from other mammalian UV-endonucleases so far described in that it seems to be dimer specific. The enzyme is stimulated by Mg2+ and is inactive in the presence of EDTA. It binds to UV-irradiated DNA-Sepharose from which it is released by low concentrations of KCl. Gel filtration data indicate that the endonuclease may belong to a high molecular weight protein or protein complex. The enzyme is very labile and freezing increases its lability.

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Polyaniline is one of the most well studied conducting polymers due to its advanced electrical, chemical, redox and morphological properties. The high conductivity of regular polyaniline, when partially oxidized and doped under acidic conditions, has been associated with the formation of unique electronic states known as polarons and bipolarons. Alternative aniline oxidation products and interesting nanotube and nanorod forms have been observed as the synthesis conditions are varied. Solid-state NMR has offered great opportunities for structural investigations and the determination of molecular dynamics in such a complex and diverse material. This review summarizes various applications of solid-state NMR techniques to polyaniline and its derivatives and the information that can be obtained by solid-state NMR.

Immunoabsorption in combination with crossed immunoelectrophoresis was used for elucidation of the antigenic architecture of the Acholeplasma laidlawii cell membrane. When cells were grown in a medium containing cholesterol and unsaturated fatty acids, the cell membranes became fragile. Membrane proteins or membrane fragments, which under some conditions seemed to be enriched in specific proteins (especially protein T2), were released into the growth medium. Therefore, most of the experiments were performed with cells grown in a cholesterol-free medium. Antigenic determinants of ectoproteins were expressed to a greater extent on the exterior face of the membrane when the cells were harvested in the early phase of the growth cycle. Furthermore, for the proteins t1a and t1b, which are membrane proteins of high molecular weight, antigenic determinants were expressed on both the exterior and the cytoplasmic faces of the membrane. Antigenic determinants of protein T4b were expressed on the cytoplasmic face of the membrane. For the flavoprotein T4a, antigenic determinants were expressed exclusively on the cytoplasmic face. All antigenic determinants of the protein T2 probably were expressed on the exterior face of the membrane, whereas no antigenic determinants of protein T3 were exposed on either face of the membrane.

Methodology advancements in NMR spectroscopy are typically classified as those involving improvements in sensitivity or resolution. This chapter focuses on the detection of two or more free induction decays during a single experiment, referred to as multiple acquisitions. Collection of multidimensional experiments requires establishing correlations to link spin systems for resonance assignments. During the process of selecting the desired coherence for spectral acquisition, unwanted coherences are often created and typically discarded using phase cycling and pulsed field gradients. A wide range of experiments have been developed that implement multiple acquisition periods in both solution and solid-state NMR spectroscopy. The chapter presents examples of such methods grouped by sample type in the form of solution or solid-state NMR applications with a focus on biomolecular NMR applications. The multiple acquisition methods increase the effective sensitivity of NMR data collection since the additional spectra do not increase the overall experimental acquisition time.