Micromechanical Study of Hyperacetylated Nucleosomes Using Single Molecule Transverse Magnetic Tweezers.

Nucleosomes are stable complexes of DNA and histone proteins that are essential for the proper functioning of the genome. These structures must be unwrapped and disassembled for processes such as gene expression, replication, and repair. Histone post-translational modifications (PTMs) are known to play a significant role in regulating the structural changes of nucleosomes. However, the underlying mechanisms by which these modifications function remain unclear. In this study, we report the results of single molecule micromanipulation experiments on DNA-protein complexes composed of hyperacetylated histone proteins using transverse magnetic tweezers. The experiments were conducted by pre-extending λ-DNA with a force less than 4 pN before introducing hyperacetylated histones into the sample chamber. The DNA shortened as the histones formed complexes with it and the nucleosome arrays were then exposed to increasing tension, resulting in quantized changes in the DNA's extension with step sizes of (integral multiples of) ~50 nm. We also compared results of experiments using PTM histones and native histones with data collected for both types of histones for the same force ranges (2-80 pN) and loading rates. Our data show that hyperacetylated nucleosomes require an unbinding force of around ~2.5 pN, which is similar to that required for native histones. Moreover, we identified clear differences between the step-size distributions of native and hyperacetylated histones and found that in contrast to tethers reconstituted with native histones, the majority of nucleosomes in tethers compacted with hyperacetylated histones underwent disassembly at forces significantly lower than 6 pN.

Alcohol: basic and translational research; 15th annual Charles Lieber &1st Samuel French satellite symposium.

The present review is based on the research presented at the symposium dedicated to the legacy of the two scientists that made important discoveries in the field of alcohol-induced liver damage: Professors C.S. Lieber and S.W. French. The invited speakers described pharmacological, toxicological and patho-physiological effects of alcohol misuse. Moreover, genetic biomarkers determining adverse drug reactions due to interactions between therapeutics used for chronic or infectious diseases and alcohol exposure were discussed. The researchers presented their work in areas of alcohol-induced impairment in lipid protein trafficking and endocytosis, as well as the role of lipids in the development of fatty liver. The researchers showed that alcohol leads to covalent modifications that promote hepatic dysfunction and injury. We concluded that using new advanced techniques and research ideas leads to important discoveries in science.

Spermidine Prevents Ethanol and Lipopolysaccharide-Induced Hepatic Injury in Mice.

To date, there is no effective treatment for alcoholic liver disease, despite its prevalence world-wide. Because alcohol consumption is associated with oxidative stress-induced liver injury and pro-inflammatory responses, naturally occurring antioxidants and/or anti-inflammatories may be potential therapeutics. Spermidine is an abundant, ubiquitous polyamine that has been found to display strong antioxidant and anti-inflammatory properties. To further investigate whether spermidine is an effective intervention for alcohol-induced liver disease, we examined its hepatoprotective properties using a two-hit, chronic ethanol and acute lipopolysaccharide (LPS)-induced mouse model of liver injury. We determined that spermidine administration prevented ethanol and LPS-induced increases in liver injury using plasma ALT as a readout. Furthermore, histological analysis of tissue from control and treated animals revealed that the pathology associated with ethanol and LPS treatment was prevented in mice additionally treated with spermidine. As predicted, spermidine also prevented ethanol and LPS-induced oxidative stress by decreasing the levels of both reactive oxygen species (ROS) and lipid peroxidation. We further determined that spermidine treatment prevented the nuclear translocation of nuclear factor κB (NFκB) by blocking the phosphorylation of the inhibitory protein, IκB, thereby preventing expression of pro-inflammatory cytokines. Finally, by measuring expression of known markers of hepatic stellate cell activation and monitoring collagen deposition, we observed that spermidine also prevented alcohol and LPS-induced hepatic fibrosis. Together, our results indicate that spermidine is an antioxidant thereby conferring anti-inflammatory and anti-fibrotic effects associated with alcoholic liver injury.

Alcohol-induced microtubule acetylation leads to the accumulation of large, immobile lipid droplets.

Although steatosis (fatty liver) is a clinically well-described early stage of alcoholic liver disease, surprisingly little is known about how it promotes hepatotoxicity. We have shown that ethanol consumption leads to microtubule hyperacetylation that can explain ethanol-induced defects in protein trafficking. Because almost all steps of the lipid droplet life cycle are microtubule dependent and because microtubule acetylation promotes adipogenesis, we examined droplet dynamics in ethanol-treated cells. In WIF-B cells treated with ethanol and/or oleic acid (a fatty acid associated with the "Western" diet), we found that ethanol dramatically increased lipid droplet numbers and led to the formation of large, peripherally located droplets. Enhanced droplet formation required alcohol dehydrogenase-mediated ethanol metabolism, and peripheral droplet distributions required intact microtubules. We also determined that ethanol-induced microtubule acetylation led to impaired droplet degradation. Live-cell imaging revealed that droplet motility was microtubule dependent and that droplets were virtually stationary in ethanol-treated cells. To determine more directly whether microtubule hyperacetylation could explain impaired droplet motility, we overexpressed the tubulin-specific acetyltransferase αTAT1 to promote microtubule acetylation in the absence of alcohol. Droplet motility was impaired in αTAT1-expressing cells but to a lesser extent than in ethanol-treated cells. However, in both cases, the large immotile droplets (but not small motile ones) colocalized with dynein and dynactin (but not kinesin), implying that altered droplet-motor microtubule interactions may explain altered dynamics. These studies further suggest that modulating cellular acetylation is a potential strategy for treating alcoholic liver disease.NEW & NOTEWORTHY Chronic alcohol consumption with the "Western diet" enhances the development of fatty liver and leads to impaired droplet motility, which may have serious deletrious effects on hepatocyte function.

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells.

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5' nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface.

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N-acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity.NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol-exposed hepatocytes and is explained by differential effects of alcohol dehydrogenase (ADH)- and cytochrome P450 2E1 (CYP2E1)-mediated ethanol metabolism on the Jak2/STAT5B pathway.

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 α6ß4 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.

Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells.

MAL2 (myelin and lymphocyte protein 2) is thought to regulate at least two steps in the hepatic apical transcytotic pathway. As vesicle budding and delivery at each step are driven by complex machineries, we predicted that MAL2 participates in several large protein complexes with multiple binding partners. To identify novel MAL2 interactors, we performed split-ubiquitin yeast two-hybrid assays and identified STK16 (serine/threonine kinase 16) as a putative interactor which we verified morphologically and biochemically. As STK16 is a Golgi-associated constitutively active kinase implicated in regulating secretion and because of the massive constitutive secretory capacity of hepatic cells, we tested whether MAL2 and STK16 function in secretion. Expression of a dominant-negative kinase-dead STK16 mutant (E202A) or knockdown of MAL2 impaired secretion that correlated with decreased expression of albumin and haptoglobin. By using 19°C temperature blocks and lysosome deacidification, we determined that E202A expression or MAL2 knockdown did not interfere with albumin synthesis or processing, but led to albumin lysosomal degradation. We conclude that MAL2 and the constitutively active STK16 function to sort secretory soluble cargo into the constitutive secretory pathway at the TGN (trans-Golgi network) in polarized hepatocytes.

Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach.

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

Alcohol-induced defects in hepatic transcytosis may be explained by impaired dynein function.

Alcoholic liver disease has been clinically well described, but the molecular mechanisms leading to hepatotoxicity have not been fully elucidated. Previously, we determined that microtubules are hyperacetylated and more stable in ethanol-treated WIF-B cells, VL-17A cells, liver slices, and in livers from ethanol-fed rats. From our recent studies, we believe that these modifications can explain alcohol-induced defects in microtubule motor-dependent protein trafficking including nuclear translocation of a subset of transcription factors. Since cytoplasmic dynein/dynactin is known to mediate both microtubule-dependent translocation and basolateral to apical/canalicular transcytosis, we predicted that transcytosis is impaired in ethanol-treated hepatic cells. We monitored transcytosis of three classes of newly synthesized canalicular proteins in polarized, hepatic WIF-B cells, an emerging model system for the study of liver disease. As predicted, canalicular delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-canalicular puncta en route to the canalicular surface that aligned along acetylated microtubules. We further determined that the stalled transcytosing proteins colocalized with dynein/dynactin in treated cells. No changes in vesicle association were observed for either dynein or dynactin in ethanol-treated cells, but significantly enhanced dynein binding to microtubules was observed. From these results, we propose that enhanced dynein binding to microtubules in ethanol-treated cells leads to decreased motor processivity resulting in vesicle stalling and in impaired canalicular delivery. Our studies also importantly indicate that modulating cellular acetylation levels with clinically tolerated deacetylase agonists may be a novel therapeutic strategy for treating alcoholic liver disease.

Lysine acetylation induced by chronic ethanol consumption impairs dynamin-mediated clathrin-coated vesicle release.

UNLABELLED: The liver is the major site of ethanol metabolism and thus sustains the most injury from chronic alcohol consumption. Ethanol metabolism by the hepatocyte leads to the generation of reactive metabolites and oxygen radicals that can readily adduct DNA, lipids, and proteins. More recently, it has become apparent that ethanol consumption also leads to increased post-translational modifications of the natural repertoire, including lysine hyperacetylation. Previously, we determined that alcohol consumption selectively impairs clathrin-mediated internalization in polarized hepatocytes. However, neither the step at which the block occurs nor the mechanism responsible for the defect have been identified. To identify the specific step at which clathrin-mediated internalization is impaired, we examined the distributions, levels, and assembly of selected components of the clathrin machinery in control and ethanol-treated cells. To determine whether the impairment is caused by ethanol-induced lysine acetylation, we also examined the same coat components in cells treated with trichostatin A (TSA), a deacetylase inhibitor that leads to protein hyperacetylation in the absence of ethanol. CONCLUSION: We determined that both ethanol and TSA impair internalization at a late stage before vesicle fission. We further determined that this defect is likely the result of decreased dynamin recruitment to the necks of clathrin-coated invaginations resulting in impaired vesicle budding. These results also raise the exciting possibility that agents that promote lysine deacetylation may be effective therapeutics for the treatment of alcoholic liver disease.

Alcohol-induced tubulin post-translational modifications directly alter hepatic protein trafficking.

BACKGROUND: Chronic ethanol exposure leads to enhanced protein acetylation and acetaldehyde adduction. Of the multitude of proteins that are modified on ethanol administration, tubulin is among the best studied. However, an open question is whether these modifications are observed in patient samples. Both modifications have also been implicated in promoting alcohol-induced defects in protein trafficking, but whether they do so directly is also unanswered. METHODS AND RESULTS: We first confirmed that tubulin was hyperacetylated and acetaldehyde-adducted in the livers from ethanol-exposed individuals to a similar extent as observed in the livers from ethanol-fed animals and hepatic cells. Livers from individuals with nonalcohol-associated fatty liver showed modest increases in tubulin acetylation, whereas nonalcohol-associated fibrotic human and mouse livers showed virtually no tubulin modifications. We also asked whether tubulin acetylation or acetaldehyde adduction can directly explain the known alcohol-induced defects in protein trafficking. Acetylation was induced by overexpressing the α-tubulin-specific acetyltransferase, αTAT1, whereas adduction was induced by directly adding acetaldehyde to cells. Both αTAT1 overexpression and acetaldehyde treatment significantly impaired plus-end (secretion) and minus-end (transcytosis)-directed microtubule-dependent trafficking and clathrin-mediated endocytosis. Each modification led to similar levels of impairment as observed in ethanol-treated cells. The levels of impairment by either modification showed no dose dependence or no additive effects suggesting that substoichiometric tubulin modifications lead to altered protein trafficking and that lysines are not selectively modified. CONCLUSIONS: These results not only confirm that enhanced tubulin acetylation is observed in human livers but that it is most relevant to alcohol-induced injury. Because these tubulin modifications are associated with altered protein trafficking that alters proper hepatic function, we propose that changing the cellular acetylation levels or scavenging free aldehydes are feasible strategies for treating alcohol-associated liver disease.

CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression-Free Survival on BTK Inhibitor Therapy.

PURPOSE: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. RESULTS: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4-mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK-STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d- CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d- were estimated progression-free at 8 years (P = 0.0004). CONCLUSIONS: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. See related commentary by Tissino et al., p. 3560.

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.

Hepatic microtubule acetylation and stability induced by chronic alcohol exposure impair nuclear translocation of STAT3 and STAT5B, but not Smad2/3.

Although alcoholic liver disease is clinically well described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we found that alcohol exposure led to increased microtubule acetylation and stability in polarized, hepatic WIF-B cells and in livers from ethanol-fed rats. Because microtubules are known to regulate transcription factor nuclear translocation and dynamic microtubules are required for translocation of at least a subset of these factors, we examined whether alcohol-induced microtubule acetylation and stability impair nuclear translocation. We examined nuclear delivery of factors representing the two mechanisms by which microtubules regulate translocation. To represent factors that undergo directed delivery, we examined growth hormone-induced STAT5B translocation and IL-6-induced STAT3 translocation. To represent factors that are sequestered in the cytoplasm by microtubule attachment until ligand activation, we examined transforming growth factor-ß-induced Smad2/3 translocation. We found that ethanol exposure selectively impaired translocation of the STATs, but not Smad2/3. STAT5B delivery was decreased to a similar extent by addition of taxol (a microtubule-stabilizing drug) or trichostatin A (a deacetylase inhibitor), agents that promote microtubule acetylation in the absence of alcohol. Thus the alcohol-induced impairment of STAT nuclear translocation can be explained by increased microtubule acetylation and stability. Only ethanol treatment impaired STAT5B activation, indicating that microtubules are not important for its activation by Jak2. Furthermore, nuclear exit was not changed in treated cells, indicating that this process is also independent of microtubule acetylation and stability. Together, these results raise the exciting possibility that deacetylase agonists may be effective therapeutics for the treatment of alcoholic liver disease.

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.

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.

Exogenous MAL reroutes selected hepatic apical proteins into the direct pathway in WIF-B cells.

Unlike simple epithelial cells that directly target newly synthesized glycophosphatidylinositol (GPI)-anchored and single transmembrane domain (TMD) proteins from the trans-Golgi network to the apical membrane, hepatocytes use an indirect pathway: proteins are delivered to the basolateral domain and then selectively internalized and transcytosed to the apical plasma membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been identified as regulators of direct and indirect apical delivery, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway.

MAL2-Induced Actin-Based Protrusion Formation is Anti-Oncogenic in Hepatocellular Carcinoma.

Recent studies report that the polarity gene myelin and lymphocyte protein 2 (MAL2), is overexpressed in multiple human carcinomas largely at the transcript level. Because chromosome 8q24 amplification (where MAL2 resides) is associated with hepatocellular- and cholangio-carcinomas, we examined MAL2 protein expression in these human carcinoma lesions and adjacent benign tissue using immunohistochemistry. For comparison, we analyzed renal cell carcinomas that are not associated with chromosome 8q24 amplification. Surprisingly, we found that MAL2 protein levels were decreased in the malignant tissues compared to benign in all three carcinomas, suggesting MAL2 expression may be anti-oncogenic. Consistent with this conclusion, we determined that endogenously overexpressed MAL2 in HCC-derived Hep3B cells or exogenously expressed MAL2 in hepatoma-derived Clone 9 cells (that lack endogenous MAL2) promoted actin-based protrusion formation with a reciprocal decrease in invadopodia. MAL2 overexpression also led to decreased cell migration, invasion and proliferation (to a more modest extent) while loss of MAL2 expression reversed the phenotypes. Mutational analysis revealed that a putative Ena/VASP homology 1 recognition site confers the MAL2-phenotype suggesting its role in tumor suppression involves actin remodeling. To reconcile decreased MAL2 protein expression in human carcinomas and its anti-oncogenic phenotypes with increased transcript levels, we propose a transcriptional regulatory model for MAL2 transient overexpression.

Alcoholic-Hepatitis, Links to Brain and Microbiome: Mechanisms, Clinical and Experimental Research.

The following review article presents clinical and experimental features of alcohol-induced liver disease (ALD). Basic aspects of alcohol metabolism leading to the development of liver hepatotoxicity are discussed. ALD includes fatty liver, acute alcoholic hepatitis with or without liver failure, alcoholic steatohepatitis (ASH) leading to fibrosis and cirrhosis, and hepatocellular cancer (HCC). ALD is fully attributable to alcohol consumption. However, only 10-20% of heavy drinkers (persons consuming more than 40 g of ethanol/day) develop clinical ALD. Moreover, there is a link between behaviour and environmental factors that determine the amount of alcohol misuse and their liver disease. The range of clinical presentation varies from reversible alcoholic hepatic steatosis to cirrhosis, hepatic failure, and hepatocellular carcinoma. We aimed to (1) describe the clinico-pathology of ALD, (2) examine the role of immune responses in the development of alcoholic hepatitis (ASH), (3) propose diagnostic markers of ASH, (4) analyze the experimental models of ALD, (5) study the role of alcohol in changing the microbiota, and (6) articulate how findings in the liver and/or intestine influence the brain (and/or vice versa) on ASH; (7) identify pathways in alcohol-induced organ damage and (8) to target new innovative experimental concepts modeling the experimental approaches. The present review includes evidence recognizing the key toxic role of alcohol in ALD severity. Cytochrome p450 CYP2E1 activation may change the severity of ASH. The microbiota is a key element in immune responses, being an inducer of proinflammatory T helper 17 cells and regulatory T cells in the intestine. Alcohol consumption changes the intestinal microbiota and influences liver steatosis and liver inflammation. Knowing how to exploit the microbiome to modulate the immune system might lead to a new form of personalized medicine in ALF and ASH.