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µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via β-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM, an important modulator of metabolism, are reviewed.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3′‐triiodo‐l‐thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ‐Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage‐specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3‐ and androgen‐mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth., What's new? Thyroid hormones may play a role in the progression of prostate cancer (PCa). In this study, the authors found that PCa cells had decreased expression of the thyroid‐hormone‐binding protein μ‐Crystallin (CRYM). Lower CRYM was also associated with poor prognosis in men with PCa. In addition, CRYM inhibited thyroid‐hormone and androgen signaling, as well as 18F‐fluoromethylcholine uptake by PCa cells. These results suggest that CRYM may act as a novel antagonist against factors that fuel PCa progression, and therefore thyroid signalling may offer a new therapeutic target in slowing aggressive prostate cancer.

Objective: To study the role of long non-coding RNA (lncRNA) CRYM-AS1 in human gastric cancer. Methods: Expression levels of CRYM-AS1 in cell lines and clinical tissues were examined by RT-qPCR. The association between CRYM-AS1 levels and clinicopathological parameters / survival rates of gastric cancer patients was analyzed.Cell functional experiments including MTT assay, glucose consumption / lactate production / ATP production detection were performed to examine the role of CRYM-AS1 in cell aerobic glycolysis and cell proliferation of gastric cancer cells. Subcellular fractionation location detection, western blot, RIP (RNA binding protein immunoprecipitation) assay, CHIP (Chromatin immunoprecipitation) assay and BSP (Bisulfite sequencing PCR) assay were carried out to explore the molecular mechanism of CRYM-AS1 in gastric cancer cells.Results: CRYM-AS1 was low expressed in gastric cancer cells and tissues compared with normal gastric cells and tissues respectively. CRYM-AS1 was negatively correlated with TNM staging, tumor size and overall survival (OS) rate in gastric cancer patients. CRYM-AS1 inhibited gastric cancer cell aerobic glycolysis and cell proliferation. CRYM-AS1 directly bound to EZH2 and mediated the CRYM promoter methylation and consequently negatively regulated the expression of CRYM. Forced expression of CRYM rescued the decreased aerobic glycolysis and cell proliferation induced by CRYM-AS1 in gastric cancer cells.Conclusion: CRYM-AS1 was an important biomarker and could be used for human gastric cancer treatment.

Background Tumor‐educated platelets (TEPs) may enable blood‐based cancer diagnosis. This study aimed to identify diagnostic TEPs genes involved in carcinogenesis. Materials and Methods The TEPs differentially expressed genes (DEGs) between healthy samples and early/advanced cancer samples were obtained using bioinformatics. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were used to identify the pathways and functional annotation of TEPs DEGs. Protein‐protein interaction of these TEPs DEGs was analyzed based on the STRING database and visualized by Cytoscape software. The correlation analysis and diagnostic analysis were performed to evaluate the diagnostic value of TEPs mRNAs expression for early/advanced cancers. Quantitative real‐time PCR (qRT‐PCR) was applied to validate the role of DEGs in cancers. Results TEPs mRNAs were mostly involved in protein binding, extracellular matrix, and cellular protein metabolic process. RSL24D1 was negatively correlated to early‐stage cancers compared to healthy controls and may be potentially used for early cancer diagnosis. In addition, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, and IFITM3 showed an upward trend in the expression from early to advanced cancer stages. Moreover, ARL2, FCGR2A, and KLHDC8B were positively associated with advanced, metastatic cancers compared to healthy controls. Among the 12 selected DEGs, the expression of 7 DEGs, including RSL24D1, IFI27, CRYM, HBD, IFITM3, FCGR2A, and KLHDC8B, were verified by the qRT‐PCR method. Conclusion This study suggests that the 7‐gene TEPs liquid‐biopsy biomarkers may be used for cancer diagnosis and monitoring., Receiving operating characteristic (ROC) curves show the relative diagnostic performance of TEPs RSL24D1, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, IFITM3, ARL2, FCGR2A, and KLHDC8B have predictive validation value for cancers.

Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

Gene mutation has an important role in disease pathogenesis; therefore, genetic screening is a useful tool for diagnosis. The present study screened pathogenic genes, ectodysplasin A (EDA) and lamin A/C (LMNA), in a patient with suspected syndromic hearing impairment and various other symptoms including tooth and skin abnormalities. Large‑scale sequencing of 438 deafness‑associated genes and whole‑genome sequencing was also performed. The present findings did not identify copy number variation and mutations in EDA; therefore, excluding the possibility of EDA‑initiated ectodermal dysplasia syndrome. A synonymous mutation in LMNA, possibly due to a splicing abnormality, did not elucidate the pathogenesis of Hutchinson‑Gilford progeria syndrome. Whole‑genome sequencing revealed copy number variations or mutations in various candidate genes which may elucidate part of the symptoms observed. The copy number variations and mutations were also used to identify single nucleotide variations (SNVs) in crystallin mu (CRYM), RAB3 GTPase activating protein catalytic subunit 1 (RAB3GAP1) and Wnt family member 10A (WNT10A), implicated in deafness, hypogonadism and tooth/skin abnormalities, respectively. The importance of an existing SNV in CRYM and a novel SNV in RAB3GAP1 in pathogenesis remains to be further elucidated. The WNT10A p.G213S mutation was confirmed to be the etiological cause of tooth agenesis and ectodermal dysplasia as previously described. It was concluded that a mutation in WNT10A may be the reason for some of the symptoms observed in the patient; however, other genes may also be involved for other symptoms. The findings of the present study provide putative gene mutations that require further investigation in order to determine their roles in pathogenesis.

Objective: Cochlear implantation is the most important treatment currently available for profound sensorineural hearing loss. The aim of this study was to investigate the etiology of hearing loss in patients with cochlear implantation, and to compare outcomes. Methods: Japanese hearing loss patients who received cochlear implants (CIs) or electric acoustic stimulation (EAS) in Shinshu University hospital (n = 173, prelingual onset: 92, postlingual onset: 81) participated in this study. Invader assay followed by the targeted exon-sequencing of 63 deafness genes using Massively parallel DNA sequencing (MPS) was applied. For prelingual patients, additional imaging examination, cCMV screening, and pediatric examination were performed for precise diagnosis. Results: Genetic screening successfully identified the causative mutation in 60% of patients with prelingual onset hearing loss and in 36% of those with postlingual hearing loss. Differences in the kinds of genes identified were observed between the two groups. Although there were marked variations in the outcome of cochlear implantation, patients with specific deafness gene mutations showed relatively good results. Conclusion: The present study showed genetic etiology is a major cause of hearing loss in CI/EAS patients. Patients possessing mutations in a number of deafness genes known to be expressed within inner ear have achieved satisfactory auditory performance, suggesting that the identification of the genetic background facilitates the prediction of post-CI performance. MPS is a powerful tool for the identification of causative deafness genes in patients receiving cochlear implantation. Therefore, determination of the involved region inside/outside of the cochlea by identification of the responsible gene is essential.

Transport of thyroid hormone (TH) across the plasma membrane is necessary for the genomic action of T3 mediated by its nuclear T3 receptor. MCT8 and MCT10 have been identified as important TH transporters. Mutations in MCT8 result in severe psychomotor retardation. In addition to TH transport into the cell, MCT8 and MCT10 also facilitate TH efflux from cells. Therefore, the aim of this study was to examine if MCT8 and MCT10 increase the availability of T3 for its nuclear receptor rather than generate a rapid equilibrium between cellular and serum T3. T3 action was investigated in JEG3 cells co-transfected with TRβ1 and a T3 response element-driven luciferase construct, and T3 metabolism was analyzed in cells transfected with type 3 deiodinase (D3). In addition, cells were transfected with MCT8 or MCT10 and/or the cytoplasmic T3-binding protein mu-crystallin (CRYM). Luciferase signal was markedly stimulated by incubating cells for 24 h with 1 nM T3, but this response was not augmented by MCT8 or MCT10 expression. Limiting the time of T3 exposure to 1-6 h and co-transfection with CRYM allowed for a modest increase in luciferase response to T3. In contrast, T3 metabolism by D3 was potently stimulated by MCT8 or MCT10 expression, but it was not affected by expression of CRYM. These results suggest that MCT8 and MCT10 by virtue of their bidirectional T3 transport have less effect on steady-state nuclear T3 levels than on T3 levels at the cell periphery where D3 is located. CRYM alters the dynamics of cellular TH transport but its exact function in the cellular distribution of TH remains to be determined.

Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ∆(1)-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ∆(1)-piperideine-2-carboxylate (P2C) and its reduced metabolite L-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to L-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [μ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3'-triiodothyronine (T3) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of -C=N- (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ∆1-piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main L-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in L-ornithine/L-glutamate/L-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.

μ-Crystallin is an NADPH-dependent cytosolic T3-binding protein. A knockout study in mice showed that μ-crystallin has a physiological function as a reservoir of T3 in the cytoplasm in vivo. Patients with nonsyndromic deafness were reported to have point mutations in the μ-crystallin gene. The expression of μ-crystallin is regulated by multiple factors. The present study was performed to determine whether thyroid function is related to the expression of μ-crystallin mRNA in peripheral mononuclear cells. We examined 23 normal healthy male and female subjects and 15 patients with Graves' disease. μ-Crystallin protein expression was determined immunohistochemically in peripheral mononuclear cells. The expression of μ-crystallin mRNA was assessed by reverse transcription of total RNA from peripheral mononuclear cells followed by quantitative PCR. μ-Crystallin protein was detected in peripheral mononuclear cells. The mRNA expression was negatively correlated with age in normal female subjects. The values in female subjects were significantly higher than those in males. The values were positively correlated with serum TSH concentration. The values of the thyrotoxic patients with Graves' disease were lower than those in healthy subjects. A transient increase in μ-crystallin expression was observed within 14-42 days after the initial treatment with antithyroid medication. Thyroid hormone inversely relates to the expression of μ-crystallin mRNA in euthyroid mononuclear cells. Abrupt suppression of thyroid function leads to overexpression of μ-crystallin mRNA in thyrotoxic mononuclear cells. Thyroid hormone-regulated μ-crystallin expression may control thyroid hormone action via the intracytoplasmic T? capacity., Article, HORMONE AND METABOLIC RESEARCH. 41(7):548-553 (2009)

http://www.elsevier.com/wps/find/journaldescription.cws_home/505783/description#description | http://www.elsevier.com/wps/find/journaldescription.cws_home/505783/description#description, Article, TRENDS IN ENDOCRINOLOGY AND METABOLISM. 18(7): 286-289

To identify proteins expressed abnormally in facioscapulohumeral muscular dystrophy (FSHD), we extracted soluble proteins from deltoid muscle biopsies from unaffected control and FSHD patients and analyzed them using two-dimensional electrophoresis, mass spectrometry and immunoblotting. Muscles from patients with FSHD showed large increases over controls in a single soluble, 34 kDa protein (pI=5.08) identified by mass spectrometry and immunoblotting as mu-crystallin (CRYM). Soluble fractions of biopsies of several other myopathies and muscular dystrophies showed no appreciable increases in mu-crystallin. Mu-crystallin has thyroid hormone and NADPH binding activity and so may influence differentiation and oxidative stress responses, reported to be altered in FSHD. It is also linked to retinal and inner ear defects, common in FSHD, suggesting that its up-regulation may play a specific and important role in pathogenesis of FSHD.

Recently, crystalized mouse ketimine reductase/CRYM complexed with NADPH was found to have pyruvate bound in its active site. We demonstrate that the enzyme binds α-keto acids, such as pyruvate, in solution, and catalyzes the formation of N-alkyl-amino acids from alkylamines and α-keto acids (via reduction of imine intermediates), but at concentrations of these compounds not expected to be encountered in vivo. These findings confirm that, mechanistically, ketimine reductase/CRYM acts as a classical imine reductase and may explain the finding of bound pyruvate in the crystallized protein.

Human cytosolic 3,5,3'-triiodo-L-thyronine-binding protein, also called mu-crystallin or CRYM, plays important physiological roles in transporting 3,5,3'-triiodo-L-thyronine (T(3)) into nuclei and regulating thyroid-hormone-related gene expression. The crystal structure of human CRYM's bacterial homolog Pseudomonas putida ornithine cyclodeaminase and Archaeoglobus fulgidus alanine dehydrogenase have been available, but no CRYM structure has been reported. Here, we report the crystal structure of human CRYM bound with NADPH refined to 2.6 A, and there is one dimer in the asymmetric unit. The structure contains two domains: a Rossmann fold-like NADPH-binding domain and a dimerization domain. Different conformations of the loop Arg83-His92 have been observed in two monomers of human CRYM in the same asymmetric unit. The peptide bond of Val89-Pro90 is a trans-configuration in one monomer but a cis-configuration in the other. A detailed comparison of the human mu-crystallin structure with its structurally characterized homologs including the overall comparison and superposition of active sites was conducted. Finally, a putative T(3)-binding site in human CRYM is proposed based on comparison with structural homologs.

This study examined whether the AR-CAG repeat length might affect clinical characteristics (testis volume) seminal parameters (sperm count and its mobility) along with hormonal serum profile [FSH, LH, Testosterone (T) and Inhibin B (InhB)] both in idiopathic male infertility (IM) and in infertility due to a previous condition of cryptorchidism (CryM) or to Y chromosome long arm microdeletions (YM).Observational study without intervention(s).One hundred and ten IM patients [90 idiopathic olizoospermic males (IOM) and 20 idiopathic azoospermic males (IAM)], 19 CryM male and 10 YM patients were included. Sixty-one age-matched healthy men who had fathered within 3 years were involved representing the control group (FM).AR-CAG repeats stretch was significantly longer in IOM (p0.05), CryM (p0.05) and YM (p0.001) than FM. When the AR-CAG repeat tracts were subdivided in three subgroups according to the length of CAG repeats tract assessed in fertile subjects (the one with the middle (n 19-21) belonging to the 25 and 75 % inter-quartile, the ends belonging to the25 % inter-quartile and75 % inter-quartile, respectively), there was a statistically significant difference of distribution of AR-CAG tract length among fertile and different groups of infertile men (p=0.0005; chi-square test). Moreover, the subgroup of AR-CAG repeat stretch with 22-28 triplets was associated with lower levels of InhB both in idiopathic oligozoospermic (Scheffe, Bonferroni and Dunett tests p=0.01) and azoospermic men (Scheffe, Bonferroni and Dunett test p=0.05), while, when FM and men with idiopathic infertility were gathered in a single group, both the subgroup of AR- CAG tract with 15-18 repeats and the one with 22-28 repeats are associated with lower testis volume, reduced sperm count and serum InhB levels.Our study showed that the outliers of AR-CAG repeat length seem to influence the function of AR, affecting testis volume and Sertoli cell function and consequently sperm production in both fertile and idiopathic infertile men.

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.

Transport of (sulfated) iodothyronines across the plasma membrane is required for their intracellular metabolism. Rat Na+/taurocholate cotransporting polypeptide (Ntcp: Slc10al) has been identified as an important transporter protein. We demonstrate that among the 7 members of the solute carrier family SLC10, only human SLC10A1 mediates sodium-dependent transport of the iodothyronine T4 and iodothyronine sulfates T3S and T4S. In contrast to SLC10A2-7, cells co-expressing SLC10A1 and the deiodinase D1 demonstrate a dramatic increase in T3S and T4S metabolism. The SLC10A1 substrates taurocholate, DHEAS and E3S inhibit T3S and T4S transport. Furthermore, co-transfection of SLC10A1 with CRYM,a well known intracellular iodothyronine-binding protein, results in an enhanced intracellular accumulation of T3S and T4S, indicating that CRYM binds iodothyronine sulfates. The present findings indicate that the liver-specific transporter SLC10A1 transports (sulfated) iodothyronines, thereby increasing their intracellular availability. Therefore, SLC10A1 may fulfill a critical step in providing liver D1 with iodothyronine sulfates for rapid degradation. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

During a search for glucose-regulated abundant mRNAs in the diabetic rat kidney, we cloned thyroid hormone binding protein (THBP), also known as μ-crystallin or CRYM. The aim of this study was to investigate the effect of hyperglycemia/high glucose on the expression of THBP. THBP mRNA copy numbers were determined in kidneys and hearts of diabetic GK rats vs normoglycemic Wistar rats, and in human mesangial cells (HMCs) exposed to high glucose using real-time qPCR, and THBP protein levels were measured by Western blotting and immunofluorescence. Intracellular ROS was measured in THBP transfected cells using DCF fluorescence. Hyperglycemia significantly increased THBP mRNA in GK rat kidneys (326 ± 50 vs 147 ± 54, p < 0.05), and hearts (1583 ± 277 vs 191 ± 63, p < 0.05). Moreover, the levels of THBP mRNA increased with age and hyperglycemia in GK rat kidneys, whereas in normoglycemic Wistar rat kidneys there was a decline with age. High glucose significantly increased THBP mRNA (92 ± 37 vs 18 ± 4, p < 0.005), and protein in HMCs. The expression of THBP as a fusion protein in transfected HMCs resulted in reduction of glucose-induced intracellular ROS. We have shown that THBP mRNA is increased in diabetic kidney and heart, is regulated by high glucose in renal cells, and appears to attenuate glucose-induced intracellular ROS. These data suggest that THBP may be involved in the cellular pathways activated in response to glucose. This is the first report linking hyperglycemia with THBP and suggests that the role of THBP in diabetic complications should be further investigated.

The crystallins were discovered more than 100 years ago by Morner (1893. Untersuchungen der Proteinsubstanzen in den lichtbrechenden Medien des Auges. Z. Physiol. Chem. 18, 61-106) as the main structural proteins of the vertebrate eye lens. Since that time the major mammalian crystallins referred to as alpha-, beta-, and gamma-crystallins were characterized with respect to their genetic organization, regulation of their expression pattern and participation in several diseases. In recent years, more and more crystallins have also been identified outside the lens. Evolutionary analysis has demonstrated the relationship of crystallins to proteins involved in protection against stress. The alpha-crystallins form large complexes up to 1Mio Da; they are built up by two subunits referred to as alphaA- and alphaB-crystallins. These subunits are encoded by individual genes, Cryaa and Cryab being localized on different chromosomes and members of the small heat-shock protein family. The alphaA-crystallin is considered to be a molecular chaperone. It is expressed mainly in the lens - mutations in the Cryaa gene lead to recessive or dominant cataracts. In contrast, the alphaB-crystallin is rather ubiquitously expressed; mutations in the Cryab gene are associated with a broad variety of neurological, cardiac and muscular disorders. The beta/gamma-crystallin super family is encoded by at least 14 genes; the proteins are characterized by four Greek key motifs. In mammals, these genes are not only organized as individual genes (Cryba1, Cryba2, Crygf, Crygs, CrygN), but also in duplets (Cryba4-Crybb1 and Crybb2-Crybb3) and in one major cluster (Cryga-Cryge). The various Cryb and Cryg genes are considered to have been evolved by various duplications of the Greek key encoding units. The two main families are distinguished by the fact that each Greek key motif in the Cryb genes is encoded by one exon, whereas two motifs are encoded by one single exon in the Cryg genes. An intermediate between these subfamilies is CrygN encoding the first two Greek key motifs by individual exons, but the others by one single exon. Mutations in the Cryb/Cryg genes lead mainly to an opacification of the eye lens. In some Cryg mutants evidence was presented that the formation of large amyloid-like intranuclear inclusions containing the altered gamma-crystallins is a key event in cataract formation. Cataract formation, caused by Cryg mutations is further characterized by stopping the secondary lens fiber differentiation as indicated by the presence of remnants of cell nuclei, which are usually degraded in secondary fiber cells. Moreover, additional clinical features are being increasingly reported since these crystallins are found outside the eye: the betaB2-crystallin (previously referred to the basic principle crystallin) is also involved in neurogenesis and male infertility. For some of the beta/gamma-crystallins, Ca(2+)-binding properties have been discussed; however, it is an unsolved question whether these crystallins serve as Ca(2+) stores in vivo. Enzyme crystallins are enzymes, which have been recruited to the lens and are expressed there in high concentrations. The mu- and zeta-crystallins (gene symbols: Crym and Cryz, respectively) are discussed as examples for mammals. Mutations in the human CRYM gene lead to non-syndromic deafness, and mutations in the Cryz gene of guinea pigs cause cataracts.

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[125I]T3, 3) a marked stimulation of cellular T4 and, particularly, T3 uptake, 4) a significant inhibition of T3 uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T4 and T3 by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein μ-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T4 and T3. In the absence of CRYM, hMCT10 and hMCT8 increased T3 uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T4 than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Research on molecular mechanisms underlying the carcinogenesis of non-small cell lung cancer (NSCLC) may provide gene targets in critical pathways valuable for improving the efficacy of therapy and survival of patients with NSCLC. However, the molecular markers highly sensitive for the prognosis and treatment evaluation of NSCLC are not yet available. To explore candidates, we conducted an oligonucleotide microarray study with three pairs of NSCLC and normal lung tissue, and determined 8 differentially expressed genes including the Human MutT homologue (hMTH1), Surfactant protein D (SPD), Human hyaluronan binding protein 2 (HABP2), Crystalline-mu (CRYM), Ceruloplasmin (CP), Integrin alpha-11 subunit (ITGA11), Collagen type XI alpha I (COL11A1), and Lung-specific X protein (Lun X). Four lung cancer-related markers MUC-1, hTERT, hnRNP B1, and CK-19 were also incorporated for further analysis. The expression profiles of the twelve genes in seventy pairs of NSCLC tumor and normal lung tissue were then detected quantitatively by using membrane array and quantitative real-time PCR (qRT-PCR). The data of the membrane array and qRT-PCR were compared for consistency and the potential of these mRNA markers in clinical application. The results showed that membrane array and qRT-PCR obtained consistent data for the tested genes in both sensitivity and specificity (correlation coefficient 0.921, p

Background: In a search for mutations of m-crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C-terminus in patients with nonsyndromic deafness. Objective: To investigate the mechanism of hearing loss caused by CRYM mutations Methods: T3 binding activity of mutant m-crystallin was compared with that of wild-type m-crystallin, because mcrystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where m-crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody. Results: One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that m-crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K-ATPase. Conclusions: CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. m-Crystallin may be involved in the potassium ion recycling system together with Na,KATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K-ATPase, T3, and deafness.