Pamela L. Tuma

ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion.

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette (ABC) transporters that limit intestinal absorption and promote biliary excretion of neutral sterols. Mutations in either ABCG5 or ABCG8 result in an identical clinical phenotype, suggesting that these two half-transporters function as heterodimers. Expression of both G5 and G8 is required for either protein to be transported to the plasma membrane of cultured cells. In this paper we used immunofluorescence microscopy to confirm, in vivo, that G5 is localized to the apical membranes of mouse enterocytes and hepatocytes. Other ABC half-transporters function as homodimers or as heterodimers with other subfamily members. To determine whether G5 or G8 complex with other ABCG half-transporters, we co-expressed G1, G2, and G4 with either G5 or G8 in cultured cells. G1, G2, and G4 co-immunoprecipitated with G5, and G4 co-immunoprecipitated with G8, but the putative dimers were retained in the endoplasmic reticulum (ER). Adenovirus-mediated expression of either G5 or G8 in the liver of G5G8 null mice resulted in ER retention of the expressed proteins and no increase in biliary cholesterol. In contrast, co-expression of G5 and G8 resulted in transit of the proteins out of the ER and a 10-fold increase in biliary cholesterol concentration. Finally, adenoviral expression of G2 in the presence or absence of G5 or G8 failed to promote sterol excretion into bile. These experiments indicate that G5 and G8 function as obligate heterodimers to promote sterol excretion into bile.

Chronic Ethanol Consumption Induces Global Hepatic Protein Hyperacetylation

BACKGROUND Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly. METHODS To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis. RESULTS In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers. CONCLUSIONS Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases.

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089–1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI(3)P, was differentially regulating endocytosis from the two domains, we examined the PI(3)P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI(3)P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI(3)P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI(3)P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.

Dynamin Forms Polymeric Complexes in the Presence of Lipid Vesicles CHARACTERIZATION OF CHEMICALLY CROSS-LINKED DYNAMIN MOLECULES

Dynamin is a GTP-binding protein that is involved in the release of coated endocytic vesicles from the plasma membrane. We have been characterizing the enzymatic properties of purified rat brain dynamin to better understand how GTP binding and hydrolysis relate to its proposed function. Previously, we have demonstrated that activation of dynamin GTPase results from positive cooperative associations between dynamin molecules as they are bound to a polymeric surface. Our present report has extended these studies and has examined the structural features of dynamin self-association. After treatment with the zero-length protein cross-linking reagent, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, dynamin in solution was found cross-linked into dimers. This homodimer likely reflects the native soluble state of the molecule. After binding to brain vesicles, dynamin was cross-linked into higher order oligomers of greater than 800 kDa. Dynamin, copurified on brain membranous organelles, also formed multimeric complexes when cross-linked suggesting dynamin exists in polymeric form in vivo. No cross-linked species other than homo-oligomers were observed, providing no evidence for close interactions between dynamin and membrane proteins. From experiments examining the effects of GTP, GDP, guanosine 5′-3-O-(thio)triphosphate, and 5′-guanylyl-β,-imidodiphosphate on cross-linking, we have determined that both dynamin membrane binding and self-association occur independently from the nucleotide-bound state of the enzyme. An 80-kDa dynamin fragment that is lacking its carboxyl-terminal domain is not cross-linked into higher order oligomers, suggesting that this domain is required for binding of dynamin to membranes and the subsequent enhancement of oligomerization. However, the dynamin fragment was found to form dimers indicating that this domain is not required for dynamin dimerization. Cross-linked dynamin was able to cooperatively bind microtubules, but did not exhibit GTPase activation. We propose that intramolecular cross-links in the dynamin monomer impart structural constraints that prevent the enhancement of GTP hydrolysis. We describe a model of the dynamin activation process to be considered in further investigations of the role for dynamin in endocytic vesicle formation.

Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking.

We have been using polarized hepatic WIF‐B cells to examine ethanol‐induced liver injury. Previously, we determined microtubules were more highly acetylated and more stable in ethanol‐treated WIF‐B cells. We proposed that the ethanol‐induced alterations in microtubule dynamics may explain the ethanol‐induced defects in membrane trafficking that have been previously documented. To test this, we compared the trafficking of selected proteins in control cells and cells treated with ethanol or with the histone deacetylase 6 inhibitor trichostatin A (TSA). We determined that exposure to 50 nM TSA for 30 minutes induced microtubule acetylation (∼3‐fold increase) and stability to the same extent as did ethanol. As shown previously in situ, the endocytic trafficking of the asialoglycoprotein receptor (ASGP‐R) was impaired in ethanol‐treated WIF‐B cells. This impairment required ethanol metabolism and was likely mediated by acetaldehyde. TSA also impaired ASGP‐R endocytic trafficking, but to a lesser extent. Similarly, both ethanol and TSA impaired transcytosis of the single‐spanning apical resident aminopeptidase N (APN). For both ASGP‐R and APN and for both treatments, the block in trafficking was internalization from the basolateral membrane. Interestingly, no changes in transcytosis of the glycophosphatidylinositol‐anchored protein, 5′‐nucleotidase, were observed, suggesting that increased microtubule acetylation and stability differentially regulate internalization. We further determined that albumin secretion was impaired in both ethanol‐treated and TSA‐treated cells, indicating that increased microtubule acetylation and stability also disrupted this transport step. Conclusion: These results indicate that altered microtubule dynamics explain in part alcohol‐induced defects in membrane trafficking. (HEPATOLOGY 2008.)

Alcohol-Induced Alterations in Hepatic Microtubule Dynamics Can Be Explained by Impaired Histone Deacetylase 6 Function

We have been using polarized, hepatic WIF‐B cells to examine ethanol‐induced liver injury. These cells polarize in culture and maintain numerous liver‐specific activities including the ability to metabolize alcohol. Previously, we found that microtubules were more highly acetylated and more stable in ethanol‐treated WIF‐B cells and that increased microtubule acetylation required ethanol metabolism and was likely mediated by acetaldehyde. This study was aimed at identifying the mechanism responsible for increased microtubule acetylation. We examined the expression of two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF‐B cells. Immunoblotting, immunofluorescence microscopy, and assays using the SirT2 inhibitor nicotinamide revealed that WIF‐B cells do not express SirT2. In contrast, HDAC6 was highly expressed in WIF‐B cells. Addition of trichostatin A (TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as in ethanol‐treated cells (approximately threefold). Although immunofluorescence labeling revealed that HDAC6 distribution did not change in ethanol‐treated cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6 solubility was increased in nocodazole‐treated cells, suggesting impaired microtubule binding. Direct microtubule binding assays confirmed this hypothesis. The decreased microtubule binding was partially prevented by 4‐methyl pyrazole, indicating the effect was in part mediated by acetaldehyde. Interestingly, HDAC6 from ethanol‐treated cells was able to bind and deacetylate exogenous tubulin to the same extent as control, suggesting that ethanol‐induced tubulin modifications prevented HDAC6 binding to endogenous microtubules. Conclusion: We propose that lower HDAC6 levels combined with decreased microtubule binding lead to increased tubulin acetylation in ethanol‐treated cells. (HEPATOLOGY 2008.)

MAL, but not MAL2, expression promotes the formation of cholesterol-dependent membrane domains that recruit apical proteins

Our recent studies have been aimed at understanding the mechanisms regulating apical protein sorting in polarized epithelial cells. In particular, we have been investigating how lipid rafts serve to sort apical proteins in the biosynthetic pathway. The recent findings that lipid domains are too small or transient to host apically destined cargo have led to newer versions of the hypothesis that invoke proteins required for lipid domain coalescence and stabilization. MAL (myelin and lymphocyte protein) and its highly conserved family member, MAL2, have emerged as possible regulators of this process in the direct and indirect apical trafficking pathways respectively. To test this possibility, we took a biochemical approach. We determined that MAL, but not MAL2, self-associates, forms higher-order cholesterol-dependent complexes with apical proteins and promotes the formation of detergent-resistant membranes that recruit apical proteins. Such biochemical properties are consistent with a role for MAL in raft coalescence and stabilization. These findings also support a model whereby hydrophobic mismatch between the long membrane-spanning helices of MAL and the short-acyl-chain phospholipids in the Golgi drive formation of lipid domains rich in raft components that are characterized by a thicker hydrophobic core to alleviate mismatch.

Alcohol consumption impairs hepatic protein trafficking: mechanisms and consequences

Alcoholic liver disease is a major biomedical health concern in the United States. Despite considerable research efforts aimed at understanding the progression of the disease, the specific mechanisms leading to alcohol-induced damage remain elusive. Numerous proteins are known to have alcohol-induced alterations in their dynamics. Defining these defects in protein trafficking is an active area of research. In general, two trafficking pathways are affected: transport of newly synthesized secretory or membrane glycoproteins from the Golgi to the basolateral membrane and clathrin-mediated endocytosis from the sinusoidal surface. Both impaired secretion and internalization require ethanol metabolism and are likely mediated by acetaldehyde. Although the mechanisms by which ethanol exposure impairs protein trafficking are not fully understood, recent work implicates alcohol-induced modifications on tubulin or components of the clathrin machinery as potential mediators. Furthermore, the physiological ramifications of impaired protein trafficking are not fully understood. In this review, we will list and discuss the proteins whose trafficking patterns are known to be impaired by ethanol exposure. We will then describe what is known about the possible mechanisms leading to impaired protein trafficking and how disrupted protein trafficking alters liver function and may explain clinical features of the alcoholic patient.

MAL2 Selectively Regulates Polymeric IgA Receptor Delivery from the Golgi to the Plasma Membrane in WIF-B cells

Myelin and lymphocyte protein 2 (MAL2) has been identified as a hepatic transcytotic regulator that mediates delivery from basolateral endosomes to the subapical compartment (SAC). However, overexpression of polymeric immunoglobulin A‐receptor (pIgA‐R) in polarized, hepatic WIF‐B cells led to the dramatic redistribution of MAL2 into the Golgi and all the transcytotic intermediates occupied by the receptor. Although overexpressed hemagglutinin and dipeptidylpeptidase IV (DPPIV) distributed to the same compartments, MAL2 distributions did not change indicating the effect is selective. Cycloheximide treatment led to decreased pIgA‐R and MAL2 intracellular staining, first in the Golgi then the SAC, suggesting they were apically delivered and that MAL2 was mediating the process. This was tested in Clone 9 cells (that lack endogenous MAL2). When expressed alone, pIgA‐R was restricted to the Golgi whereas when coexpressed with MAL2, it distributed to the surface, was internalized and delivered to MAL2‐positive puncta. In contrast, DPPIV distributions were independent of MAL2. Surface delivery of newly synthesized pIgA‐R, but not DPPIV, was enhanced greater than ninefold by MAL2 coexpression. In WIF‐B cells where MAL2 expression was knocked down, pIgA‐R, but not DPPIV, was retained in the Golgi and its basolateral delivery was impaired. Thus, in addition to its role in transcytosis, MAL2 also regulates pIgA‐R delivery from the Golgi to the plasma membrane.

UV exposure modulates hemidesmosome plasticity, contributing to long-term pigmentation in human skin

Human skin colour, ie pigmentation, differs widely among individuals, as do their responses to various types of ultraviolet radiation (UV) and their risks of skin cancer. In some individuals, UV‐induced pigmentation persists for months to years in a phenomenon termed long‐lasting pigmentation (LLP). It is unclear whether LLP is an indicator of potential risk for skin cancer. LLP seems to have similar features to other forms of hyperpigmentation, eg solar lentigines or age spots, which are clinical markers of photodamage and risk factors for precancerous lesions. To investigate what UV‐induced molecular changes may persist in individuals with LLP, clinical specimens from non‐sunburn‐inducing repeated UV exposures (UVA, UVB or UVA + UVB) at 4 months post‐exposure (short‐term LLP) were evaluated by microarray analysis and dataset mining. Validated targets were further evaluated in clinical specimens from six healthy individuals (three LLP+ and three LLP−) followed for more than 9 months (long‐term LLP) who initially received a single sunburn‐inducing UVA + UVB exposure. The results support a UV‐induced hyperpigmentation model in which basal keratinocytes have an impaired ability to remove melanin that leads to a compensatory mechanism by neighbouring keratinocytes with increased proliferative capacity to maintain skin homeostasis. The attenuated expression of SOX7 and other hemidesmosomal components (integrin α6β4 and plectin) leads to increased melanosome uptake by keratinocytes and points to a spatial regulation within the epidermis. The reduced density of hemidesmosomes provides supporting evidence for plasticity at the epidermal–dermal junction. Altered hemidesmosome plasticity, and the sustained nature of LLP, may be mediated by the role of SOX7 in basal keratinocytes. The long‐term sustained subtle changes detected are modest, but sufficient to create dramatic visual differences in skin colour. These results suggest that the hyperpigmentation phenomenon leading to increased interdigitation develops in order to maintain normal skin homeostasis in individuals with LLP. Copyright