BBA - Molecular Basis of Disease (v.1792, #10)

Diabetes and the peripheral nerve by Irina G. Obrosova (931-940).
Diabetes-induced damage to peripheral nerve culminates in development of peripheral diabetic neuropathy (PDN), one of the most devastating complications of diabetes mellitus and a leading cause of foot amputation. The pathogenesis of PDN occurs as a consequence of complex interactions among multiple hyperglycemia-initiated mechanisms, impaired insulin signaling, inflammation, hypertension, and disturbances of fatty acid and lipid metabolism. This review describes experimental new findings in animal and cell culture models as well as clinical data suggesting the importance of 1) previously established hyperglycemia-initiated mechanisms such as increased aldose reductase activity, non-enzymatic glycation/glycooxidation, activation of protein kinase C, 2) oxidative–nitrosative stress and poly(ADP-ribose) polymerase activation; 3) mitogen-activated protein kinase and cyclooxygenase-2 activation, impaired Ca++ homeostasis and signaling, and several other mechanisms, in PDN.
Keywords: Diabetic neuropathy; Aldose reductase; Non-enzymatic glycation; Oxidative–nitrosative stress; Protein kinase C; Poly(ADP-ribose) polymerase; Mitogen-activated protein kinase; Nuclear factor-κB; Cyclooxygenase-2; 12/15-lipoxygenase; Calcium signaling;

Phospholipase A2 subclasses in acute respiratory distress syndrome by Eirini Kitsiouli; George Nakos; Marilena E. Lekka (941-953).
Phospholipases A2 (PLA2) catalyse the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids. In acute lung injury-acute respiratory distress syndrome (ALI-ARDS) several distinct isoenzymes appear in lung cells and fluid. Some are capable to trigger molecular events leading to enhanced inflammation and lung damage and others have a role in lung surfactant recycling preserving lung function: Secreted forms (groups sPLA2-IIA, -V, -X) can directly hydrolyze surfactant phospholipids. Cytosolic PLA2 (cPLA2-IVA) requiring Ca2+ has a preference for arachidonate, the precursor of eicosanoids which participate in the inflammatory response in the lung. Ca2+-independent intracellular PLA2s (iPLA2) take part in surfactant phospholipids turnover within alveolar cells. Acidic Ca2+-independent PLA2 (aiPLA2), of lysosomal origin, has additionally antioxidant properties, (peroxiredoxin VI activity), and participates in the formation of dipalmitoyl-phosphatidylcholine in lung surfactant. PAF-AH degrades PAF, a potent mediator of inflammation, and oxidatively fragmented phospholipids but also leads to toxic metabolites. Therefore, the regulation of PLA2 isoforms could be a valuable approach for ARDS treatment.
Keywords: ALI; ARDS; Phospholipase A2; PLA2; PAF-AH; Lung surfactant; Inhibitors of PLA2;

The emerging role of TGF-β superfamily coreceptors in cancer by Carmelo Bernabeu; Jose M. Lopez-Novoa; Miguel Quintanilla (954-973).
The transforming growth factor β (TGF-β) signaling pathway plays a key role in different physiological processes such as development, cellular proliferation, extracellular matrix synthesis, angiogenesis or immune responses and its deregulation may result in tumor development. The TGF-β coreceptors endoglin and betaglycan are emerging as modulators of the TGF-β response with important roles in cancer. Endoglin is highly expressed in the tumor-associated vascular endothelium with prognostic significance in selected neoplasias and with potential to be a prime vascular target for antiangiogenic cancer therapy. On the other hand, the expression of endoglin and betaglycan in tumor cells themselves appears to play an important role in the progression of cancer, influencing cell proliferation, motility, invasiveness and tumorigenicity. In addition, experiments in vitro and in vivo in which endoglin or betaglycan expression is modulated have provided evidence that they act as tumor suppressors. The purpose of this review was to highlight the potential of membrane and soluble forms of the endoglin and betaglycan proteins as molecular targets in cancer diagnosis and therapy.
Keywords: Transforming growth factor-beta receptor; Cancer; Endoglin; Betaglycan; Angiogenesis;

A novel mutation (F71L) in αA-Crystallin with defective chaperone-like function associated with age-related cataract by S.G. Bhagyalaxmi; PNBS Srinivas; Kelly A. Barton; K. Ravi Kumar; M. Vidyavathi; J. Mark Petrash; G. Bhanuprakash Reddy; T. Padma (974-981).
Age-related cataract (ARC) is a multifactorial disease and the leading cause of blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. However, gene mutation studies on ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA (αA-crystallin) gene in three unrelated female sporadic cases among 711 ARC patients but not in 265 normal non-cataractous controls by SSCP and RFLP analysis. By comparing human recombinant wild-type and F71L-αA-crystallin, we characterized the functional significance of this missense mutation. Chromatography, fluorescence and far- and near-UV CD studies indicated that F71L missense mutation did not significantly affect the apparent molecular mass, secondary and tertiary structures and hydrophobicity of αA-crystallin. While the mutant αA-crystallin displayed significant (35–90%) loss of chaperone-like activity (CLA) in thermal aggregation of carbonic anhydrase, βL- and γ-crystallins, it showed moderate (10–50%) loss in CLA in DTT-induced aggregation of insulin and lysozyme. This is the first report of an αA-F71L mutation being associated with ARC and suggests that ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.
Keywords: Age-related cataract; αA-crystallin; F71L-mutation; SSCP; RFLP; Chaperone-like activity; CD spectra; Hydrophobicity;

Impact of β-galactosidase mutations on the expression of the canine lysosomal multienzyme complex by Robert Kreutzer; Mihaela Kreutzer; Adrian C. Sewell; Somporn Techangamsuwan; Tosso Leeb; Wolfgang Baumgärtner (982-987).
β-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the β-galactosidase gene cause the lysosomal storage disease GM1-gangliosidosis. In order to identify additional molecular changes associated with the presence of β-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1 +/+, GLB1 +/− and GLB1 −/− fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total β-galactosidase, β-galactosidase variants and protective protein for β-galactosidase gene (PPGB) in GLB1 +/− and GLB1 −/− compared to GLB1 +/+ fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant β-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1 +/+, GLB1 +/− and GLB1 −/− genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.
Keywords: Differential expression; β-galactosidase variant; Protective protein/cathepsin A; Neuraminidase 1; Lysosomal multienzyme complex; GM1-gangliosidosis;

Rhomboid domain containing 2 (RHBDD2): A novel cancer-related gene over-expressed in breast cancer by M.C. Abba; E. Lacunza; M.I. Nunez; A. Colussi; M. Isla-Larrain; A. Segal-Eiras; M.V. Croce; C.M. Aldaz (988-997).
In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n  = 46) and immunohistochemistry (n  = 213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p  = 0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n  = 17) or breast benign lesions (n  = 16) (p  = 0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p  = 0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p  = 0.0023), relapse-free survival (p  = 0.0013), and metastasis-free interval (p  = 0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.
Keywords: RHBDD2; Gene expression profile; Gene amplification; Breast cancer;

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation by Ryuichi Harada; Natsuki Kobayashi; Jihoon Kim; Chikashi Nakamura; Sung-Woong Han; Kazunori Ikebukuro; Koji Sode (998-1003).
Human α-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of α-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of α-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in α-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form α-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.
Keywords: Parkinson's disease; α-Synuclein; Fibrillation; E46K; Imperfect repeat sequences;

Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD) by Kristen J. Skvorak; Elizabeth J. Hager; Erland Arning; Teodoro Bottiglieri; Harbhajan S. Paul; Stephen C. Strom; Gregg E. Homanics; Qin Sun; Erwin E.W. Jansen; Cornelis Jakobs; William J. Zinnanti; K. Michael Gibson (1004-1010).
Skvorak et al. demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.
Keywords: Hepatocyte transplantation (HTx); Monoamine; Leucine; Large neutral amino acid; GABA; Intracellular turnover;

Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide by Toshikazu Shioiri; Masashi Muroi; Fumihiko Hatao; Masato Nishida; Toshihisa Ogawa; Yoshikazu Mimura; Yasuyuki Seto; Michio Kaminishi; Ken-ichi Tanamoto (1011-1018).
Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h  following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.
Keywords: Apoptosis; Toll-like receptor; Sepsis; Endotoxin; Infection;

Transient receptor potential vanilloid (TRPV) 1 channels function as sensors for a variety of noxious and inflammatory signals, including capsaicin, heat and protons, and are up-regulated under inflammatory conditions. As end-stage kidney disease (ESKD) is associated with chronic inflammation, impaired immunity and depressed lymphocyte numbers, we sought to determine whether altered TRPV1 (and related TRPV2) expression in immune cells might be a contributing factor. TRPV1 and TRPV2 mRNA expression in peripheral blood mononuclear cells (PBMC) was similar in controls and ESKD patients by quantitative real-time RT-PCR. However, using immunocytochemistry, TRPV1-immunoreactivity was significantly higher and TRPV2-immunoreactivity was significantly lower in PBMC from ESKD patients compared to controls. The plant-derived TRPV1 agonists, capsaicin and resiniferatoxin (RTX) and the putative endovanilloid/endocannabinoids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA), induced concentration-dependent death of PBMC from healthy donors with a rank order of potency of RTX > NADA > OLDA >> capsaicin. TRPV1 (5′-iodoresiniferatoxin) and cannabinoid (CB2; AM630) receptor antagonists blocked the cytotoxic effect of NADA. In subsequent experiments, PBMC from ESKD patients exhibited significantly increased susceptibility to NADA-induced death compared to PBMC from controls. The apparent up-regulation of TRPV1 may be a response to the inflammatory milieu in which PBMC exist in ESKD and may be responsible for the increased susceptibility of these cells to NADA-induced death, providing a possible explanation as to why ESKD patients have reduced lymphocyte counts and impaired immune function. Thus, TRPV1 (and possibly CB2) antagonists may have potential for the treatment of immune dysfunction in ESKD.
Keywords: Cell death; End-stage kidney disease; Mononuclear cells; N-arachidonoyl-dopamine; TRPV1; TRPV2;

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration by Jian Jiao; Kaihua Sun; Will P. Walker; Pooneh Bagher; Christina D. Cota; Teresa M. Gunn (1027-1035).
Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a “PSAP” motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.
Keywords: TSG101; Spongiform neurodegeneration; Mahogunin Ring Finger-1; MGRN1; Ubiquitination; Endocytic trafficking;

Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia by Jitendra Badhai; Anne-Sophie Fröjmark; Edward J. Davey; Jens Schuster; Niklas Dahl (1036-1042).
Diamond–Blackfan anemia (DBA) is a severe congenital anemia characterized by a specific decrease of erythroid precursors. The disease is also associated with growth retardation, congenital malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. We show herein that primary fibroblasts from DBA patients with truncating mutations in RPS19 or in RPS24 have a marked reduction in proliferative capacity. Mutant fibroblasts are associated with extended cell cycles and normal levels of p53 when compared to w.t. cells. RPS19 mutant fibroblasts accumulate in the G1 phase, whereas the RPS24 mutant cells show an altered progression in the S phase resulting in reduced levels in the G2/M phase. RPS19 deficient cells exhibit reduced levels of Cyclin-E, CDK2 and retinoblastoma (Rb) protein supporting a cell cycle arrest in the G1 phase. In contrast, RPS24 deficient cells show increased levels of the cell cycle inhibitor p21 and a seemingly opposing increase in Cyclin-E, CDK4 and CDK6. In combination, our results show that RPS19 and RPS24 insufficient fibroblasts have an impaired growth caused by distinct blockages in the cell cycle. We suggest this proliferative constraint to be an important contributing mechanism for the complex extra-hematological features observed in DBA.
Keywords: RPS19; RPS24; Diamond–Blackfan anemia; Proliferation; Cell cycle; Cell cycle regulation;