Vimentin, a major constituent of the intermediate filament family of proteins, is ubiquitously expressed in normal mesenchymal cells and is known to maintain cellular integrity and provide resistance against stress. Vimentin is overexpressed in various epithelial cancers, including prostate cancer, gastrointestinal tumors, tumors of the central nervous system, breast cancer, malignant melanoma, and lung cancer. Vimentin’s overexpression in cancer correlates well with accelerated tumor growth, invasion, and poor prognosis; however, the role of vimentin in cancer progression remains obscure. In recent years, vimentin has been recognized as a marker for epithelial–mesenchymal transition (EMT). Although EMT is associated with several tumorigenic events, vimentin’s role in the underlying events mediating these processes remains unknown. By virtue of its overexpression in cancer and its association with tumor growth and metastasis, vimentin serves as an attractive potential target for cancer therapy; however, more research would be crucial to evaluate its specific role in cancer. Our recent discovery of a vimentin-binding mini-peptide has generated further impetus for vimentin-targeted tumor-specific therapy. Furthermore, research directed toward elucidating the role of vimentin in various signaling pathways would reveal new approaches for the development of therapeutic agents. This review summarizes the expression and functions of vimentin in various types of cancer and suggests some directions toward future cancer therapy utilizing vimentin as a potential molecular target.

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Vimentin in cancer and its potential as a molecular target for cancer therapy.

Reactive oxygen species (ROS) are well known for their role in mediating both physiological and pathophysiological signal transduction. Enzymes and subcellular compartments that typically produce ROS are associated with metabolic regulation, and diseases associated with metabolic dysfunction may be influenced by changes in redox balance. In this review, we summarize the current literature surrounding ROS and their role in metabolic and inflammatory regulation, focusing on ROS signal transduction and its relationship to disease progression. In particular, we examine ROS production in compartments such as the cytoplasm, mitochondria, peroxisome, and endoplasmic reticulum and discuss how ROS influence metabolic processes such as proteasome function, autophagy, and general inflammatory signaling. We also summarize and highlight the role of ROS in the regulation metabolic/inflammatory diseases including atherosclerosis, diabetes mellitus, and stroke. In order to develop therapies that target oxidative signaling, it is vital to understand the balance ROS signaling plays in both physiology and pathophysiology, and how manipulation of this balance and the identity of the ROS may influence cellular and tissue homeostasis. An increased understanding of specific sources of ROS production and an appreciation for how ROS influence cellular metabolism may help guide us in the effort to treat cardiovascular diseases.

Reactive Oxygen Species in Metabolic and Inflammatory Signaling.

Crystallins in the eye: Function and pathology.

Modern classification of the family of human small heat shock proteins (the so-called HSPB) is presented, and the structure and properties of three members of this family are analyzed in detail. Ub...

Large Potentials of Small Heat Shock Proteins

Small heat shock proteins: Role in cellular functions and pathology.

Congenital cataract is a common major abnormality of the eye, which frequently causes blindness in children. It is clinically and genetically heterogeneous, and the phenotype varies considerably between and within families.1 It is most commonly inherited in an autosomal dominant manner, although recessive and X-linked forms also exist.2–4 Congenital cataract may be isolated, be associated with other ocular developmental abnormality, or be a part of systemic disease. To date, mutations in dozens of genes have been described in association with isolated congenital cataract, including members of the crystallin and connexin families,5 membrane proteins MIP6 and LIM2,2 cytoskeletal protein BFSP2,7,8 and transcription factors PITX39 and HSF4.10

Crystallins account for nearly 90% of total lens protein and, not surprisingly, mutations in the crystallin genes represent a large proportion of the mutations identified in autosomal dominant congenital cataract (ADCC). These include αA-crystallin (CRYAA).2,11 αB-crystallin (CRYAB),12 βA1/A3-crystallin (CRYBA1/A3),13,14 βB1-crystallin (CRYBB1),15 βB2-crystallin (CRYBB2),16,17 γC-crystallin (CRYGC),18,19 and γD-crystallin (CRYGD) mutations.18,20 Among the lens crystallins, α-crystallin is considered to play an important role in maintaining the transparency of the lens. α-Crystallins are members of the highly conserved small heat shock protein (sHsp) family. A typical mammalian lens contains approximately 35% α-crystallin, which makes it one of the major protein components that produce the necessary refractive index. α-Crystallin in the lens exists as hetero-oligomers composed of two components, αA- and αB-crystallin. The subunits of αA- and αB-crystallin have molecular masses of ∼20 kDa with approximately 57% amino-acid sequence homology. The ratio of αA to αB in the lens is generally 3:1. In addition to being a major structural building block, α-crystallin, like other members of the sHsp family, has chaperone-like activity in preventing aggregation of other lens proteins induced by heat or other stressors.21–24

Two mutations in the CRYAB gene have been reported to be associated with cataract. A missense mutation R120G was associated with cataract and desmin-related myopathy,25 and a deletion mutation, 450delA, was associated with isolated autosomal dominant posterior polar cataract.12 In the present study, we report a novel D140N mutation in the CRYAB gene in an ADCC family with isolated lamellar cataract. To characterize the possible structural and functional changes that underlie the molecular mechanism of cataract caused by this mutation, we expressed the wild-type and the mutant αB-crystallin in vitro, and compared the secondary, tertiary, and quaternary structures as well as chaperone-like activity between the wild-type and mutant proteins.

A Novel αB-Crystallin Mutation Associated with Autosomal Dominant Congenital Lamellar Cataract

Purpose Missense mutations in crystallin genes have been identified in autosomal dominant congenital cataracts. A truncation in the CRYBB2 gene (Q155*) has been associated with cerulean cataract, however its effects on biophysical properties have not been reported. We sought to determine the changes in conformation and protein-protein interactions brought about by this mutation. Methods Site specific mutations were performed to obtain the Q155* betaB2-crystallin mutant. Protein-protein interactions were screened by a mammalian two-hybrid system assay. Conformational changes were studied with spectroscopy (circular dichroism and fluorescence) and FPLC chromatography. Results We detected a decrease in protein-protein interactions for the Q155* betaB2-crystallin mutant. The Q155* mutant shows decreased ordered structure and stability but the partially unfolded protein retains some dimer structure. Conclusions The Q155* mutation in betaB2-crystallin causes changes in biophysical properties that might contribute to cataract formation.

Interaction and biophysical properties of human lens Q155* betaB2-crystallin mutant.

Purpose The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract. Methods Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor. The lens vimentin gene was fused into a GFP vector and the alphaB-crystallin (WT or R120G mutant) gene was fused into the RFP vector. The donor-acceptor plasmid pairs of intermediate filament (IF)-GFP and alphaB-RFP were co-transfected into HeLa cells. After incubation, confocal fluorescence images of the transfected cells were taken. FRET was estimated by the acceptor photobleaching method. Protein-protein interaction was evaluated by FRET efficiency. Results The confocal fluorescence images showed that the cells expressing vimentin and R120G alphaB-crystallin contained large amounts of protein aggregates while few vimentin fibers were observed. FRET efficiency analyses indicated that vimentin had a significantly greater protein-protein interaction with R120G alphaB-crystallin than with WT alphaB-crystallin. Conclusions Our results show that the R120G alphaB-crystallin mutant promoted vimentin aggregation through increased protein-protein interaction. This process may contribute to the formation of congenital cataract.

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Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

Lens fiber cells contain high concentrations of proteins in the cytoplasm, which gives the lens a high refractive index and minimizes light-scattering at the membrane-cytoplasm interface. α-Crystallins are the major proteins of the mammalian lens that constitute approximately 35% of its dry weight and exist as large heterogeneous oligomers composed of two types of subunits, αA-crystallin and αB-crystallin, in a molar ratio of 3 to 1 in most mammalian lenses.1 The two subunits have molecular masses of ~20 kDa with approximately 57% amino-acid sequence homology. In humans, αA-crystallin contains 173 amino acids and αB-crystallin contains 175.2,3 Both subunits are also expressed in other tissues.1,4 In addition to being a major structural protein, α-crystallins have a chaperone-like activity that suppresses protein aggregation or promotes the refolding of unfolded proteins. Thus, α-crystallins play important roles in maintaining the transparency of the lens.1,5–7

Protein aggregation and precipitation is one of the causes of lens opacification. Studies indicate that calpain-mediated cleavage of lens crystallins plays an important role in the aggregation and precipitation of lens proteins.8–11 This unregulated cleavage of crystallins may result in the precipitation of β-crystallins or cytoskeletal proteins12–14 or reduction of chaperone activity of α-crystallins.15 Calpains belong to a superfamily of structurally related, calcium-activated cysteine proteases.16–18 Some calpains are ubiquitously expressed, whereas others are tissue specific.17,19–21 Studies of various cataract animal models have suggested that Lp82 and calpain-2 may be the major calpains involved in murine cataractogenesis.10,11,22–28

Both α- and β-crystallins, but not γ-crystallins, are susceptible to calpain-mediated cleavage.29 Whereas α-crystallins are cleaved by calpains at the C terminus,30 β-crystallins are cleaved closer to the N terminus.31 C-terminal cleavage of αA-crystallin by calpains can occur at several sites. The major cleaved products of αA-crystallin in the lens include αA1–151, αA1–156, αA1–163, and αA1–168.30 αA1–162 can be generated by incubating αA-crystallin with m-calpain in vitro,30 but αA1–162 is barely detectable in normal lenses.30,32 However, αA1–162 is readily detected in diabetic cataractous lenses.32 The accumulation of truncated αA-crystallins in cataractous lenses could result from an increase in production and/or a decrease in degradation of the truncated products by other proteases.

The ubiquitin-proteasome pathway (UPP) is one of the proteolytic systems that selectively degrade modified or damaged proteins. We have demonstrated in previous studies that lens cells (both lens epithelial cells and lens fiber cells) have a fully functional UPP33–38 and that the UPP preferentially degrades damaged or modified proteins, including oxidized, glutathiolated and thermally denatured proteins.37,39–42 To investigate the role of the UPP in degradation of C-terminal cleaved αA-crystallins, we compared the susceptibility of wt and the C-terminal truncated αA-crystallins to UPP-mediated degradation.

The data presented indicate that C-terminal truncation of αA-crystallin significantly altered its susceptibility to UPP-mediated degradation. Whereas the susceptibility of αA1–163 to UPP-mediated degradation was similar to that of wild-type (wt) αA-crystallin, αA1–168 was less susceptible and the αA1–162 more susceptible than wt αA-crystallin to UPP-mediated degradation. The rapid degradation of αA1–162 by the UPP may explain why αA1–162-crystallin is barely detectable in the normal lenses. Because the αA1–162-crystallin is less thermally stable and prone to aggregation, the timely degradation of the αA1–162-crystallin may prevent its accumulation and aggregation in the lens.

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Degradation of C-terminal Truncated αA-crystallins by the Ubiquitin–Proteasome Pathway

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only α-crystallin or some specific γ-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (αA-, αB-, βB2-, or γC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from αA- and αB-crystallin to βB2- and γC-crystallin. Competitive interaction study using untagged αA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and αA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm. J. Cell. Biochem. 104: 51–58, 2008. © 2007 Wiley-Liss, Inc.

Bing-Fen Liu

Papers

A Novel αB-Crystallin Mutation Associated with Autosomal Dominant Congenital Lamellar Cataract

Interaction and biophysical properties of human lens Q155* betaB2-crystallin mutant.

Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

Degradation of C-terminal Truncated αA-crystallins by the Ubiquitin–Proteasome Pathway

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells.