Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).     

Modulation of Hyaluronan Synthase Activity in Cellular Membrane Fractions

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1β, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.Hyaluronan (HA)³ is the only non-sulfated linear polymer belonging to the family of glycosaminoglycans (GAGs). HA is an unbranched polymer of alternating GlcNAc and GlcUA residues linked by alternate β(1→4) and β(1→3) bonds. Native HA is typically larger than other GAGs, reaching molecular mass values between 10⁶ and 10⁷ Da.HA is a major component of extracellular matrices and in pericellular spaces, particularly in tissues with rapid cell proliferation and cell migration (1). Through interactions with cell surface receptors, notably CD44 and RHAMM (receptor for HA-mediated motility), HA has important roles in regulating cell behavior, including signal transduction, cell adhesion, proliferation, migration, and differentiation (2). Recently, novel interactions involving HA and Toll-like receptors 4 and 2 have been described that have important roles in inflammation (3, 4). Moreover, HA has been implicated in morphogenesis (5–8), wound healing (9), angiogenesis (10), malignancies, cancer growth, and tumor invasion (11).In mammals, HA is normally synthesized at the plasma membrane and extruded directly into the extracellular space by three isoforms of HA synthases (HASs), HAS1, -2, and -3. The three HAS isoforms differ in tissue distribution, regulation, and enzymatic properties (12); nevertheless, they are similar in amino acid sequences and molecular structures.HA biosynthesis is under the control of a wide variety of cytokines and growth factors (13). The changes in HA synthesis can be related to HAS mRNA expression (14), to availability of the UDP-sugar precursors (15, 16), or to modulation by phosphorylation of HAS (17–19) in response to cytokines and growth factors. Moreover, HA chain synthesis can be controlled by additional mechanisms, such as cell type, intracellular environment, or HAS accessory proteins (20). Cultures of smooth muscle cells isolated from human colon increase synthesis of HA after treatment with a viral mimetic molecule (poly(I-C)) (21). The HA is organized into novel cable-like structures, and their synthesis may be initiated in the perinuclear and/or the endoplasmic reticulum (ER) membranes (22). Furthermore, HA interstitial deposition is correlated with inflammatory processes (23, 24) in which HA-CD44 interactions stimulate leukocyte adhesion in order to generate an inflammatory response (25).Cytokines and growth factors, such as IL-1β and platelet-derived growth factor BB (PDGF-BB), as well as 4-methylumbeliferone (4-MU) and the tumor promoter phorbol 12-myristate 13-acetate (PMA), also modulate HA synthesis (26–29). In order to elucidate how these different effectors affect HAS activity, it is important to purify and solubilize the HAS enzymes as previously underlined in studies on eukaryotic cell lines (30). In this context, Itano and Kimata (31) used a mammalian transient expression system to characterize the three different HAS isoforms in either cells or cellular membrane extracts. On the other hand, Spicer (32) described three relatively simple procedures for the detection of HA synthase activity in cultured mammalian cell lines. In all of these studies, the enzyme activity was measured by incubating cellular membrane extracts with radiolabeled UDP-sugar precursors, and the final analysis of the products was done by liquid scintillation counting. Various other strategies and methods can be used to determine the HA biosynthetic capacity of cells, although they are always based on the use of radiolabeled UDP-sugar precursors (33–35).In our previous studies, we described methods of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) and high performance liquid chromatography (HPLC) for the analysis of disaccharides derived from HA and chondroitin sulfate (33–38). In order to improve the sensitivity of this method, a derivatization with 2-aminoacridone (AMAC) was done, followed by fluorescence detection (39). In this study, we modified this method to address the question of localization of HAS activity during intracellular trafficking, since HA has been detected inside cells in previous studies (40–42). This new non-radioactive method was used to quantify HAS activity on cell membranes fractionated by sucrose gradient methods. To test the robustness of our approach, we analyzed the effect of 4-MU, PMA, IL-1β, PDGF-BB, and tunicamycin on cell cultures. In particular, we found that tunicamycin induced an increase of HA synthesis in both plasma and internal cell membranes in EVC cells, whereas it increased HA synthesis only in the internal cell membranes in the OVCAR-3 cells. The results suggest that post-translational modulation of HAS activity is responsible for the increased HA synthesis inside the cells. Moreover, since tunicamycin induced HA cable structures in the OVCAR-3 cells, we correlated the altered intracellular HAS activity with the capability to promote HA cable formation.     

pH-Dependent Changes in Photoaffinity Labeling Patterns of the H1 Influenza Virus Hemagglutinin by Using an Inhibitor of Viral Fusion

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66–76, 1996, and J. Virol. 71:4062–4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the α-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.Influenza virus contains a lipid envelope that must fuse with host cell membranes in order to initiate virus infection (42, 43, 49). The hemagglutinin (HA) protein, a trimeric glycoprotein embedded in the viral membrane, is responsible for specific binding to cell surface sialic acid-containing receptors (46) and for the fusion of the two membranes (51). Although the mechanism of viral fusion is not fully elucidated, it is known that the fusion event is preceded by a conformational change occurring in the HA trimer that is triggered by the decreasing pH encountered during endosomal passage of the virus (23, 43, 49, 50). The HA trimer is composed of three identical monomers, each containing two protein subunits (designated HA1 and HA2) attached to each other via a disulfide linkage (36, 52). These monomer subunits are formed from a single chain precursor HA (HA0) that undergoes cleavage during transport from the Golgi to the cell surface (27). Entry of the influenza virus into host cells is facilitated through receptor binding by the HA1 subunit to the sialic acid-containing receptor. The conformational change brought on by the low pH of the endosome exposes the hydrophobic amino terminus of the HA2 subunit, which is believed to be a trigger in the fusion process (8, 17, 19, 40). It is postulated that the native state of the HA is a spring-loaded coiled coil and upon acidification, the hydrophobic fusion peptide is translocated toward the target membrane (9–11). This exposed hydrophobic amino terminus is believed to mediate fusion with the cell membrane (8, 19).Influenza virus HA can be cleaved from viral membrane surfaces with bromelain protease to create a soluble form of the protein (bromelain-cleaved HA [BHA]) (5, 52). The soluble HA remains a trimer with properties identical to those of the native membrane bound protein (44). Upon acidification, BHA undergoes a conformational change and forms rosettes caused by the aggregation of the exposed hydrophobic fusogenic domains of the HA2 subunit (14, 40). In this conformation, the BHA is susceptible to trypsin digestion, while it is resistant to this protease in its native conformation (15, 40).We have previously reported on the identification of a class of compounds that can inhibit influenza virus fusion (29, 30). These compounds are able to inhibit the low pH induced conformational change in the HA protein of H1 and H2 subtype viruses but not of the H3 subtype virus. Of these three subtypes, precise structural information is available only for H3 HA (8, 20, 37, 38, 45, 48). Previously a model of H1 HA was constructed using H3 HA crystal structure data (52) and a potential fusion inhibitor-binding pocket was identified within HA2 based on resistant mutation analysis and inhibitor selectivity (30). In order to probe this binding model and better understand the mechanism of action of these compounds, experiments were carried out with isolated H1 BHA. Various analogs were able to protect BHA from protease digestion following acid treatment and subsequent neutralization. A radiolabeled analog which possessed a photoactivatable azide moiety was synthesized (16). Affinity labeling at a neutral or acidic pH produced very different profiles of labeled amino acids, although in each case the amino acids were in or near the proposed binding pocket in the HA2. The consequences of the differences in HA2 photoaffinity labeling patterns with regard to the mechanism of action of these fusion inhibitors are discussed below.     

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13–1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.Extracellular vesicles (EVs)¹ are membrane surrounded structures that exist in all body fluids and all cells studied so far release EVs (1). They are heterogeneous, spherical organelles spanning between 30 to more than 1000 nm in diameter and include exosomes, microvesicles, and apoptotic bodies (2). There is increasing evidence supporting the important role of EVs in cell-to-cell communication by their delivery of proteins, lipids, and nucleic acids from one donor cell to many target cells. The generation of exosomes/prostasomes is a complicated process involving two invagination sessions of biological membranes. The first one comprises the plasma membrane contributing with endocytic vesicles in the formation of early endosomes that mature into late endosomes. The second one starts multiple inward buddings of the late endosomal membrane creating intraluminal vesicles (ILVs) therewith completing formation of multivesicular bodies (MVBs) or storage vesicles (3) thus retaining selected molecules from the maternal cell. Ceramide can induce such formation of small microdomains into larger domains (4). Ceramide is one of two cleavage products of sphingomyelin by sphingomyelinase, the other is phosphocholine (5) and prostasomes contain sphingomyelinase (6). The membrane of MVBs (storage vesicles) may fuse with the plasma membrane of the secretory cell and, in case of prostate epithelial cells, release the intraluminal vesicles as prostasomes to the extracellular space (7, 8). It is noteworthy that the bilayered membrane surrounding prostasomes (after the two sessions of invaginations) should be regarded as “right-side-out” with reference to the plasma membrane. This is illustrated by e.g. Mg²⁺ and Ca²⁺ -stimulated ATPase that is an ectoenzyme (9) that is also appearing at the outer surface of prostasomes (10). The corollary is that cell surface interactive molecules like enzymes and receptors may appear also on the membranes of exosomes/prostasomes.The majority of prostasomes ranges in diameter-size from 30–200 nm, with a mean of 142 nm (11). The main purpose of prostasomes may be to transfer newly synthesized proteins from the prostate gland to sperm and thereby, among other things, render them protection in the female genital tract (12, 13). Prostasomal proteins may be transferred to sperm through different mechanisms, viz direct interaction with the sperm membrane (14), fusion at a lowered pH (15), and internalization (16). Prostasomes are immunosuppressive and regulate the complement system and they have proven antioxidant and antibacterial properties (17, 18). Prostasomes contain a surrounding lipid membrane bilayer that exhibits a high cholesterol/phospholipid ratio (19). The lipid composition of the membrane is unusual and among the phospholipids sphingomyelin is the dominant one, contrary to other cell membranes where phosphatidylcholine is most abundant. Prostasomes have a strong contribution of saturated and monounsaturated fatty acids (19, 20). These characteristics together with a high cholesterol/phospholipid ratio make the membrane of the prostasome very stable as demonstrated by electron spin resonance (19).In the early 1970s the plasma membrane of the cell was described as a fluid mosaic by Singer and Nicholson (21), but as early as in 1953 Palade claimed that in the bilayered lipid membrane, proposed by Davson and Danielli (22), were areas of different composition, so called caveolae (23). These caveolae are invaginations of the plasma membrane (24). The first hypothesis of lipid rafts (specialized membrane domains enriched in glycosphingolipids, proteins and cholesterol) was brought up in 1988 by van Meer and Simons (25) and was subsequently elaborated in 1997 by Simons and Ikonen (26). Lipid rafts were defined as low density subdomains of the plasma membrane that are resistant to nonionic detergents at a low temperature (27, 28). Fatty acids present in lipid rafts are more saturated, compared with the membrane adjacent to the domains. It means that the fatty acids can be packed more densely and this may lead to phase separation. The abundance of intercalating cholesterol makes the rafts more rigid and less fluid than the rest of the plasma membrane (29). In other words, the membrane can undergo phase separation into co-existing liquid-disordered and liquid-ordered phases. The liquid-ordered phase (the lipid raft) becomes enriched in cholesterol and saturated fatty acids and is characterized by tight lipid packing and reduced molecular diffusion, as we noticed for prostasomes (19).There are two different types of lipid rafts, planar and caveolae. The distinguishing factor is that the caveolae are formed by the protein caveolin whereas the planar rafts lack this protein (30). Instead they contain the protein flotillin (31). Researchers have found that selected proteins localize, and colocalize in lipid rafts (32). Lipid rafts are not anchored at a specific site in the plasma membrane, but float freely. This enables larger and more stable platform domains to aggregate (33). The formed aggregates are involved in many biological functions including endocytosis, cell communication, molecular trafficking, neurotransmission and they could be understood as organizing centers for signaling molecules and receptors (30, 31). When cells are depleted of cholesterol, e.g. by the agent methyl-β-cyclodextrin, formation of caveolae expression and also raft-dependent endocytosis are inhibited (34). This demonstrates the importance of these cholesterol-enriched domains to cell survival. Flotillins are also involved in endocytosis in a process controlled by the phosphorylation of tyrosine residues (35).In this work we asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Accordingly, we prepared lipid rafts from human prostasomes in order to characterize their protein content.     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.The basement membrane is a specialized extracellular matrix (ECM)² composed of collagen, fibronectin, perlecan, agrin, and laminin. Several studies have focused on the involvement of these ECM molecules in the formation and maturation of neuromuscular junctions (1–4) and interneuronal synapses (5). More recently, much effort has been made by our group and others to understand the role of these molecules at the interface of astroglia and blood vessels (6–8). Laminin is highly expressed at the perivascular ECM, and the laminin receptor, dystroglycan (α-DG), together with many other components of the dystrophin-associated protein (DAP) complex, is particularly enriched at astrocyte endfeet abutting the blood vessels (9–11). The binding of laminin to α-DG at these specialized astrocyte domains in brain plays a key role in the polarized distribution of components of the DAP complex (6, 12).Multiple lines of evidence indicate that the DAP complex is crucial for the functional distribution both of the water-permeable channel, AQP4, and the inwardly rectifying potassium channel, Kir4.1, at astrocyte endfeet. Indeed, mutations in the dystrophin gene, deletion of α-syntrophin, or loss of laminin binding to α-DG caused by a mutation in the Large1 glycosyltransferase result in a dramatic reduction of the expression of AQP4 and Kir4.1 at perivascular astrocyte endfeet (6, 7, 12–15). The mislocalization of AQP4 in the dystrophin mutant and α-syntrophin null mice results in delayed onset of brain edema and K⁺ clearance (16–18). Collectively, these studies highlight a cooperative role of the ECM and both the extracellular and cytoplasmic components of the DAP complex in the proper targeting of proteins to functional domains of astrocytes leading to the regulation of electrolyte balance and fluid movement.Although the role of DG in targeting other members of the DAP complex (6) as well as AQP4 and Kir4.1 to astrocyte endfeet has been well established (12), the mechanisms underlying this highly organized distribution remain poorly understood. In C2C12 myotubes, agrin triggers AChR clustering, a DG-dependent process, through the coalescence of lipid rafts, which is necessary for proper AChR gating functions (19–21). In oligodendrocytes, laminin induces the relocalization of α6β1 integrin to lipid rafts containing PDGFαR, thereby providing a potential mechanism for the incorporation of cell survival signals (22). Lipid rafts are defined as small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. These small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (23). Indeed, the immunological synapse is a good example where rafts are brought together to form large functional membrane domains (24). At the immunological synapse, agrin induces the clustering of lipid rafts and their colocalization with CD3 and CD28 complex surface antigens as well as with Lck tyrosine kinase leading to T cell activation (24). Together, these studies provide evidence for a functional role of ligand-induced clustering of lipid rafts.We have previously shown that laminin induces the coclustering of the DAP complex with Kir4.1 and AQP4 in glial cell cultures (8, 25). Moreover, in vivo studies have shown that the perivascular localization of these channels and several components of the DAP complex at astrocyte endfeet require the interaction of laminin with α-DG (6, 12). In light of these data we asked whether lipid rafts contribute to the laminin-DG-dependent compartmentalization of the DAP complex and AQP4 to key active domains of astrocytes. We show here using fluorescently labeled cholera toxin subunit B (CtxB), a common marker for GM1-containing lipid rafts, that laminin induces a dramatic reorganization of GM1 into large clusters or macrodomains that colocalize extensively with components of the DAP complex in cortical astrocyte cultures. Laminin-mediated clustering of AQP4 is dependent both on cholesterol-sensitive lipid rafts and the DAP complex bringing novel insight into ECM-dependent membrane domain organization and the mechanisms underlying the polarized distribution of these proteins in astrocytes.     

Activated Dectin-1 Localizes to Lipid Raft Microdomains for Signaling and Activation of Phagocytosis and Cytokine Production in Dendritic Cells

Lipid rafts are plasma membrane microdomains that are enriched in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-anchored proteins and play an important role in the signaling of ITAM-bearing lymphocyte antigen receptors. Dectin-1 is a C-type lectin receptor (CLR) that recognizes β-glucan in the cell walls of fungi and triggers signal transduction via its cytoplasmic hemi-ITAM. However, it is not known if similar to antigen receptors, Dectin-1 would also signal via lipid rafts and if the integrity of lipid raft microdomains is important for the physiological functions mediated by Dectin-1. We demonstrate here using sucrose gradient ultracentrifugation and confocal microscopy that Dectin-1 translocates to lipid rafts upon stimulation of dendritic cells (DCs) with the yeast derivative zymosan or β-glucan. In addition, two key signaling molecules, Syk and PLCγ2 are also recruited to lipid rafts upon the activation of Dectin-1, suggesting that lipid raft microdomains facilitate Dectin-1 signaling. Disruption of lipid raft integrity with the synthetic drug, methyl-β-cyclodextrin (βmD) leads to reduced intracellular Ca²⁺ flux and defective Syk and ERK phosphorylation in Dectin-1-activated DCs. Furthermore, βmD-treated DCs have significantly attenuated production of IL-2, IL-10, and TNFα upon Dectin-1 engagement, and they also exhibit impaired phagocytosis of zymosan particles. Taken together, the data indicate that Dectin-1 and perhaps also other CLRs are recruited to lipid rafts upon activation and that the integrity of lipid rafts is important for the signaling and cellular functions initiated by this class of innate receptors.Cell membranes are dynamic and laterally inhomogeneous bi-layered structures that are composed of multiple lipid and glycoprotein species, and located within cellular membranes are specialized detergent-resistant microdomains known as lipid rafts (1). Lipid rafts are envisaged as partially ordered membrane domains caused by the close packing of glycosylphosphatidylinositol-anchored proteins with glycosphingolipids and cholesterol (2, 3). These lipid raft microdomains have been demonstrated to play an important role in facilitating the signaling of some transmembrane receptors, such as Fc receptors (4–6), cytokine receptors (7–9), and lymphocyte antigen receptors, namely the B-cell receptor (BCR)³ and T-cell receptor (TCR) (10–13). More recently, it was found that lipid rafts were also involved in the signal transduction of some innate pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). For instance, TLR2 and TLR4 have been shown to translocate to lipid rafts upon their stimulation with specific agonists (14–16). However, it is currently not known if other classes of membrane-bound PRRs are also recruited to lipid rafts and if the integrity of lipid raft microdomains is important for the cellular functions initiated by these innate receptors.Dectin-1 or Clec7a is a member of the C-type lectin receptor (CLR) family and functions as a pattern-recognition receptor by binding β-glucan found in the cell walls of pathogenic fungi such as Candida albicans (17–19). It is expressed mainly on innate cells such as macrophages, neutrophils, and dendritic cells (DCs) and plays an important role in anti-fungal immunity (17, 20, 21). Dectin-1 has been shown to mediate the phagocytosis of yeast and yeast-derived particles such as zymosan, and it could also activate the production of inflammatory cytokines in macrophages and DCs upon binding its ligands (18, 22–25).Stimulation of Dectin-1 by zymosan or its specific ligand β-glucan activates Syk tyrosine kinase (23, 24) and leads to the subsequent activation of NFκB via the Card9-Bcl10-Malt1 complex (24, 26). Recently, we demonstrated that stimulation of Dectin-1 also activated phospholipase Cγ2 (PLCγ2) and led to the induction of Ca²⁺ flux and activation of NFκB in DCs (27). Dectin-1 possesses an immunotyrosine-activated motif (ITAM) in its cytoplasmic tail (23, 24), suggesting that it is capable of mediating its own signal transduction, and more importantly, could signal in a manner analogous to the BCR and TCR. However, unlike the classical ITAM found in lymphocyte antigen receptors that comprises two tandem YXXL (where X is any amino acid) sequences, Dectin-1 has only a single YXXL and is frequently referred to as possessing a “hemi-ITAM.”Given that Dectin-1 and BCR share some downstream signaling molecules such as Syk and PLCγ2 but differ in the structure of their ITAMs, we wonder if Dectin-1 would signal via lipid rafts in a manner similar to BCR. In this report, we show that Dectin-1 as well as its downstream signaling molecules, Syk and PLCγ2, translocate to the lipid rafts upon stimulation of DCs with zymosan or β-glucan. And more importantly, we demonstrate that the integrity of the lipid raft is important for Dectin-1 to induce Ca²⁺ flux and activate phagocytosis and cytokine production in DCs upon its activation.     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.Toll-like receptors (TLRs)³ are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1, 2).Lipopolysaccharide (LPS) from Gram-negative bacteria is the ligand for the TLR4 complex (3), whereas, TLR2 can recognize lipoproteins/lipopeptides of Gram-positive bacteria and mycoplasma (1, 2). LPS forms a complex with LPS-binding protein in serum leading to the conversion of oligomeric micelles of LPS to monomers, which are delivered to CD14. Monomeric LPS is known to bind TLR4/MD-2/CD14 complex (4). Lipid A, which possesses most of the biological activities of LPS, is acylated with hydroxy saturated fatty acids. The 3-hydroxyl groups of these saturated fatty acids are further 3-Ο-acylated by saturated fatty acids. Removal of these Ο-acylated saturated fatty acids from Lipid A not only results in complete loss of endotoxic activity, but also makes Lipid A act as an antagonist against the native Lipid A (5, 6). One or more Lipid As containing unsaturated fatty acids are known to be non-toxic and act as an antagonist against endotoxin (7, 8). In addition, deacylated lipoproteins are unable to activate TLR2 and to induce cytokine expression in monocytes (9). Together, these results suggest that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. Indeed, it is suggested that the rapid interaction of bacterial lipopeptides with plasma membrane of macrophages occurs via insertion of their acylated saturated fatty acids as determined by electron energy loss spectroscopy and freeze-fracture techniques (10, 11). TLR2 can form a heterodimer with TLR1 or TLR6, which can discriminate the molecular structure of triacyl or diacyl lipopeptides (12–14). So far there is no evidence that microbial ligands for other TLRs are acylated by saturated fatty acids.Results from our previous studies demonstrated that saturated fatty acids activate TLR4 and polyunsaturated fatty acids (PUFA) inhibit both saturated fatty acid- and LPS-induced activation of TLR4 (15, 16). In addition, the saturated fatty acid lauric acid potentiates, but the n-3 PUFA docosahexaenoic acid (DHA) inhibits lipopeptide (TLR2 agonist)-induced TLR2 activation (17). Together, these results suggest that both TLR2 and TLR4 signaling pathways and target gene expression are reciprocally modulated by saturated and polyunsaturated fatty acids. However, the mechanism for this modulation by fatty acids is not understood.TLR4 is recruited to lipid raft factions after cells are treated with LPS and subsequently induces tumor necrosis factor-α expression in RAW264.7 cells (18). This process occurs in an ROS-dependent manner because inhibition of NADPH oxidase suppresses TLR4 recruitment to lipid rafts (19). Methyl-β-dextrin, a lipid raft inhibitor, significantly inhibits the LPS-induced expression of cytokine (19), suggesting that lipid rafts are essential for TLR4-mediated signal transduction and target gene expression. Lipid rafts are a collection of lipid membrane microdomains characterized by insolubility in non-ionic detergents. Lipid rafts serve as a platform where receptor-mediated signal transduction is initiated (20). Lipid rafts have a special lipid composition that is rich in cholesterol, sphingomyelin, and glycolipids (21). The polar lipids in detergent-resistant membrane contain predominantly saturated fatty acyl residues with underrepresented PUFAs (22–24), suggesting that saturated fatty acyl chains favor lipid raft association. On the other hand, n-3 PUFAs displace signaling proteins from lipid rafts by altering lipid composition, and the displacement leads to the suppression of T-cell receptor-mediated signaling (25). It is now well documented that TLRs form homo- or hetero-oligomers (1, 2). TLR4 homodimerization is the initial step of the receptor activation. Results from our previous studies suggest that the molecular target by which saturated fatty acids and n-3 PUFAs reciprocally modulate TLR4 activation is the receptor complex itself or the event leading to the receptor activation instead of the downstream signaling components (15, 16). Therefore, we determined whether the reciprocal modulation of TLR4 activation is mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and if these processes occur in an ROS-dependent manner.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction,Molecular Transport,and Callose Biosynthesis

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.The plasma membrane (PM)¹ is considered as one of the most interactive and dynamic supramolecular structures of the cell (1, 2). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4–8). Typically, PM proteins are either embedded in the phospholipid bilayer through transmembrane helices or less tightly bound to the membrane through reversible or irreversible surface interactions. In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9, 10). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9, 10). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9–11). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10, 11). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM.Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13–15), Arabidopsis (16), barrel clover (Medicago truncatula) (17), rice (18), oat, and rye (19). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15–19). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15–19). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells.Although several reports describe the partial characterization of DRMs from higher plants (13–23), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]