Mallory bodies: Lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Summary Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or α1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.     

Identification of Mallory bodies with rhodamine B fluorescence and other stains for keratin

Rhodamine B staining in conjunction with fluorescence microscopy is shown to demonstrate Mallory bodies. Mallory body morphology, localization, and distribution in hepatocytes from griseofulvin-fed mice, human hepatoma, and human alcoholics were similar to those observed in the same tissues after conventional staining methods for Mallory bodies. The presence of these inclusions was further confirmed by specific cytochemical localization with indirect immunoperoxidase labeling, horseradish peroxidase labeling, and electron microscopy. Other tinctorial or histochemical procedures previously used for keratin or prekeratin (modified Mallory stain, Kreyberg method, Pauly method for histidine) also stained Mallory bodies for study with white light microscopy but with decreasing sensitivity respectively. Mallory bodies from mouse and human liver both appear to contain a keratin-like moiety. This entity may be simply, rapidly, and permanently stained with rhodamine B, and selectively and reproducibly demonstrated with fluorescence microscopy.     

On the three-dimensional structure of quick-frozen hepatic Mallory bodies with special reference to the appearance of cytoplasmic vesicles.

Livers containing Mallory bodies (MBs, hyalin degenerative cytoplasmic inclusions) were examined using Heuser's and Van Harreveld's cryo-techniques. The tissues were collected from 1) a patient suffering from alcoholic hepatitis and 2) mice treated with griseofulvin (GF, an anti-mitotic drug). Normal mouse liver and isolated MBs from GF-treated mice were also analyzed by the same methods. Our results suggest that under the toxic influence of alcohol or GF on microtubular elements, MBs are generated by entanglement of elements of 10 nm filaments with microtubule elements. This in turn inhibits cellular transport processes. The reticular net of the ER-element which is usually observable in the normal tissue is changed into numerous small vesicles in the pathological and experimental tissues. The diameters of hepatocytes containing these vesicles were 1.5 to 2 times larger than control diameters. MBs have previously been described in thin sections as filamentous tangles. On replicas we found that they appear to be composed of pairs of filaments twisted in a roughly helical manner, each having a diameter less than 10 nm. The paired helical nature of the MB-filaments is reminiscent of other inclusion bodies, which are also composed of elements of 10 nm filaments, observable in various neurological diseases.     

Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation

Mallory bodies represent cytokeratin-rich inclusion bodies which occur characteristically but not exclusively in human alcoholic liver disease and experimentally in mice during chronic intoxication with drugs. We report the first in vitro cell system of Mallory body induction. In clone 9 rat hepatocytes stably transfected to express an ER-retained T126M-aquaporin-2 (AQP2), on the mean 40% of the cells contained cytokeratin-rich inclusion bodies. By electron microscopy, their structure corresponded to that of genuine Mallory bodies. Such inclusion bodies were not detectable in clone 9 rat hepatocytes stably expressing a Golgi apparatus/lysosome-retained E258K-aquaporin-2. Proteasome inhibition increased the number of Mallory body-containing T126M-AQP2-expressing clone 9 hepatocytes to 60% on average. Proteasome inhibition in non-transfected, cytokeratin meshwork-forming clone 9 hepatocytes resulted in Mallory body formation on average in 6% of cells. Collectively, these data suggest that in the described in vitro cell system, Mallory body formation is induced by the presence of non-native protein conformers and point to the involvement of the proteasomal digestive system. The here reported in vitro system will be useful in studies about the biogenesis and progression of Mallory bodies, their relationship to aggresomes, and the role of inclusion bodies in the pathogenesis of cell damage.     

Wheat proteins improve cryopreservation of rat hepatocytes

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze–thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance‐associated proteins such as WCS120, TaTIL, WCS19, and TaIRI‐2. These improved procedures enhance long‐term storage (2–12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77–93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non‐toxic, natural products that do not require animal serum, and are economical and easy to prepare. Biotechnol. Bioeng. 2009;103: 582–591. © 2009 Wiley Periodicals, Inc.     

Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting

Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles. The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation. We have partially purified internal vesicles and analysed their content. Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p). All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery. We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane. Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain. In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner. Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles.     

Refolding of proteins from inclusion bodies: rational design and recipes

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.     

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm,Bombyx mori

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects.     

Albumin storage proteins in the protein bodies of castor bean

Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of closely related albumins localized in the matrix of the organelle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds.