Toward unraveling the complexity of simple epithelial keratins in human disease

Omary, M. Bishr; Ku, Nam Su; Strnad, Pavel; Hanada, Shinichiro

doi:10.1172/jci37762

Cited by 243 publications

(378 citation statements)

References 127 publications

(157 reference statements)

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“…Hyperphosphorylated ubiquitin-conjugated K8/18 are the major constituents of Mallory bodies, the inclusion bodies in hepatocytes (51,52). The occurrence and formation of keratin aggregates is linked either to their phosphorylation status or to the cellular levels of K8 but not K18 (53). Interestingly, stable expression of K18-GM but not K18-WT led to its accumulation as intracellular aggregates (Fig.…”

Section: Discussionmentioning

confidence: 99%

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Budnar¹,

Vaidya

Kalraiya

2010

Journal of Biological Chemistry

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Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.

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Section: Discussionmentioning

confidence: 99%

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Budnar¹,

Vaidya

Kalraiya

2010

Journal of Biological Chemistry

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“…Stage Liver Disease-Several mutations in K8, including G62C, R341H, and G434S, predispose their carriers to acute and chronic liver disease, based on animal and human studies (16,17,38). The most common amino acid-altering K8 mutations in white and black individuals are R341H and G434S, respectively.…”

Section: Hypersumoylation Of K8 Variants That Predispose To Endmentioning

confidence: 99%

Keratin Hypersumoylation Alters Filament Dynamics and Is a Marker for Human Liver Disease and Keratin Mutation

Snider

Weerasinghe

Iñiguez‐Lluhí³

et al. 2011

Journal of Biological Chemistry

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Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/ K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.

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“…Together with actin filaments and microtubules they form a sophisticated composite network, which has been identified as a main player in cell mechanics (1). By contrast to actin filaments and microtubules, which are conserved across cell types and organisms, IFs comprise a large family of proteins, encoded by 70 genes in humans (2), and they are expressed in a cell-type-specific manner. Vimentin is an IF protein expressed in fibroblasts, the eye lens, and cells of mesenchymal origin.…”

mentioning

confidence: 99%

Lateral association and elongation of vimentin intermediate filament proteins: A time-resolved light-scattering study

Lopez

Saldanha

Huber

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Vimentin intermediate filaments (IFs) are part of a family of proteins that constitute one of the three filament systems in the cytoskeleton, a major contributor to cell mechanics. One property that distinguishes IFs from the other cytoskeletal filament types, actin filaments and microtubules, is their highly hierarchical assembly pathway, where a lateral association step is followed by elongation. Here we present an innovative technique to follow the elongation reaction in solution and in situ by time-resolved static and dynamic light scattering, thereby precisely capturing the relevant time and length scales of seconds to minutes and 60-600 nm, respectively. We apply a quantitative model to our data and succeed in consistently describing the entire set of data, including particle mass, radius of gyration, and hydrodynamic radius during longitudinal association.I ntermediate filaments (IFs) constitute one of the three protein filament systems in the cytoskeleton of metazoa. Together with actin filaments and microtubules they form a sophisticated composite network, which has been identified as a main player in cell mechanics (1). By contrast to actin filaments and microtubules, which are conserved across cell types and organisms, IFs comprise a large family of proteins, encoded by 70 genes in humans (2), and they are expressed in a cell-type-specific manner. Vimentin is an IF protein expressed in fibroblasts, the eye lens, and cells of mesenchymal origin. The monomers with a molecular weight M w of 53.5 × 10 3 g/mol share their tripartite structure consisting of an α-helical rod flanked by intrinsically disordered "head" and "tail" domains, as shown in Fig. 1A, with all other IFs. These monomers are stable in denaturing conditions, such as 8 M urea, and assemble into coiled-coil dimers and subsequently into antiparallel tetramers with M w = 214 × 10 3 g/mol, a length of 60 nm, and a diameter of 5 nm upon stepwise dialysis into low-salt buffers, such as 2 mM sodium phosphate (3). Thus, in buffer conditions without urea, these tetramers with a mass per unit length M tet L of 3,570 g/(mol·nm) are the smallest subunits and starting precursors for vimentin IF assembly. In vitro the assembly of tetramers into full-length filaments can be initiated by the addition of, e.g., monovalent salts such as potassium chloride (KCl) at concentrations of a few tens of millimolars. It has been shown by time-lapse electron microscopy (4) and more recently by real-time small-angle X-ray scattering (SAXS) in combination with microfluidic techniques (5-7) that a lateral assembly step into unit-length filaments (ULFs) consisting of typically eight tetramers (Fig. 1B) is followed by an elongation reaction where ULFs and short filaments join to form micrometer-long filaments (Fig. 1C). However, the exact molecular mechanism of the elongation reaction remains elusive. It is clear that the tail domains are not needed (4). The way that the IF consensus domains at either end of the rod interact, overlapping (8) vs. interdigitating (9), is n...

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Toward unraveling the complexity of simple epithelial keratins in human disease

Cited by 243 publications

References 127 publications

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Keratin Hypersumoylation Alters Filament Dynamics and Is a Marker for Human Liver Disease and Keratin Mutation

Lateral association and elongation of vimentin intermediate filament proteins: A time-resolved light-scattering study

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