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BBA - Molecular Basis of Disease (v.1822, #4)

Editorial Board (pp. i).

Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation by Johanna Nilsson; Adnan Halim; Ali-Reza Moslemi; Anders Pedersen; Jonas Nilsson; Göran Larson; Anders Oldfors (pp. 493-499).

Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-¹⁴C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.► The molecular pathogenesis of a new disease with glycogen depletion in muscle. ► Thr83Met substitution in glycogenin-1 abolished the autoglucosylation on Tyr 195. ► Autoglucosylation was studied in vitro with various mutated glycogenin molecules. ► The glucosylation was analyzed with nano-LC/MS/MS, western blot and radiography. ► Co-expression of various mutants of glycogenin-1 resulted in inter-glucosylation.

Keywords: Glycogen; Glycogenin; Glycogen storage disease; Cell-free expression; Mass spectrometry

Hypoxic preconditioning increases iron transport rate in astrocytes by Lei Yang; Ming Fan; Fang Du; Qi Gong; Zheng Gang Bi; Zhou Jing Zhu; Ling Ling Zhu; Ya Ke (pp. 500-508).

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.► HP leads to an increase in expression of HIF-1α and iron transporters in astrocytes. ► HP increases Tf-Fe and NTBI uptake and iron release in astrocytes. ► HP leads to a progressive increase in cellular iron content. ► HP has the ability to increase iron transport rate in astrocytes. ► The increased iron transport rate is associated with HP-induced neuroprotection.

Keywords: Hypoxic preconditioning (HP); Neuroprotection; Transferrin receptor (TfR1); Divalent metal transporter 1 (DMT1); Ferroportin1 (Fpn1); Hypoxia-inducible factor-1 alpha (HIF-1alpha)

δ-Catenin promotes E-cadherin processing and activates β-catenin-mediated signaling: Implications on human prostate cancer progression by Hangun Kim; Yongfeng He; Ilhwan Yang; Yan Zeng; Yonghee Kim; Young-Woo Seo; Mary Jo Murnane; Chaeyong Jung; Jae-Hyuk Lee; Jeong-Joon Min; Dong-Deuk Kwon; Kyung Keun Kim; Qun Lu; Kwonseop Kim (pp. 509-521).

δ-Catenin binds the juxtamembrane domain of E-cadherin and is known to be overexpressed in some human tumors. However, the functions of δ-catenin in epithelial cells and carcinomas remain elusive. We found that prostate cancer cells overexpressing δ-catenin show an increase in multi-layer growth in culture. In these cells, δ-catenin colocalizes with E-cadherin at the plasma membrane, and the E-cadherin processing is noticeably elevated. E-Cadherin processing induced by δ-catenin is serum-dependent and requires MMP- and PS-1/γ-secretase-mediated activities. A deletion mutant of δ-catenin that deprives the ability of δ-catenin to bind E-cadherin or to recruit PS-1 to E-cadherin totally abolishes the δ-catenin-induced E-cadherin processing and the multi-layer growth of the cells. In addition, prostate cancer cells overexpressing δ-catenin display an elevated total β-catenin level and increase its nuclear distribution, resulting in the activation of β-catenin/LEF-1-mediated transcription and their downstream target genes as well as androgen receptor-mediated transcription. Indeed, human prostate tumor xenograft in nude mice, which is derived from cells overexpressing δ-catenin, shows increased β-catenin nuclear localization and more rapid growth rates. Moreover, the metastatic xenograft tumor weights positively correlate with the level of 29kD E-cadherin fragment, and primary human prostate tumor tissues also show elevated levels of δ-catenin expression and the E-cadherin processing. Taken together, these results suggest that δ-catenin plays an important role in prostate cancer progression through inducing E-cadherin processing and thereby activating β-catenin-mediated oncogenic signals.► δ-Catenin overexpression is frequently observed in prostate cancer. ► We examined changes in E-cadherin stability by δ-catenin overexpression. ► δ-Catenin overexpression promotes E-cadherin processing and activates β-catenin-mediated nuclear signaling. ► Our data suggest that δ-catenin plays a role in prostate cancer progression.

Keywords: Abbreviations; PS-1; Presenilin-1; MMP; Matrix Metalloproteinase; HGF; Hepatocyte Growth Factor; CTF; C-Terminal FragmentProstate cancer; Catenin; LEF-1; E-cadherin; Androgen receptor; Wnt

Potentiation of dietary restriction-induced lifespan extension by polyphenols by Daniel J. Aires; Graham Rockwell; Ting Wang; Jennifer Frontera; Jo Wick; WenFang Wang; Marija Tonkovic-Capin; Jianghua Lu; Lezi E; Hao Zhu; Russell H. Swerdlow (pp. 522-526).

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF+PAO). Both IF and IF+PAO groups outlived the control group and the IF+PAO group outlived the IF group (all p<0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.► Polyphenol supplements can potentiate dietary restriction-induced life extension. ► Dietary restriction can induce inflammatory and oxidative stress. ► Polyphenols may mitigate dietary restriction-induced stress. ► Even with a high fat diet, alternate day feeding extends mouse lifespan.

Keywords: Longevity; Dietary restriction; Polyphenol; Tea; Blueberry; Pomegranate

Adiponectin deficiency: Role in chronic inflammation induced colon cancer by Arpit Saxena; Alexander Chumanevich; Emma Fletcher; Bianca Larsen; Kirby Lattwein; Kamaljeet Kaur; Raja Fayad (pp. 527-536).

Adiponectin (APN), an adipokine, exerts an anti-inflammatory and anti-cancerous activity with its role in glucose and lipid metabolism and its absence related to several obesity related malignancies including colorectal cancer. The aim of this study is to determine the effect of APN deficiency on the chronic inflammation-induced colon cancer. This was achieved by inducing inflammation and colon cancer in both APN knockout (KO) and C57B1/6 wild type (WT) mice. They were divided into four treatment groups (n=6): 1) control (no treatment); 2) treatment with three cycles of dextran sodium sulfate (DSS); 3) weekly doses of 1,2-dimethylhydrazine (DMH) (20mg/kg of mouse body weight) for twelve weeks; 4) a single dose of DMH followed by 3 cycles of DSS (DMH+DSS). Mice were observed for diarrhea, stool hemoccult, and weight loss and were sacrificed on day 153. Tumor area and number were counted. Colonic tissues were collected for Western blot and immunohistochemistry analyses. APNKO mice were more protected than WT mice from DSS induced colitis during first DSS cycle, but lost this protection during the second and the third DSS cycles. APNKO mice had significantly severe symptoms and showed greater number and larger area of tumors with higher immune cell infiltration and inflammation than WT mice. This result was further confirmed by proteomic study including pSTAT3, pAMPK and Cox-2 by western blot and Immunohistochemistry. Conclusively, APN deficiency contributes to inflammation-induced colon cancer. Hence, APN may play an important role in colorectal cancer prevention by modulating genes involved in chronic inflammation and tumorigenesis.► APNKO mice showed greater protection in the 1st DSS cycle but lost in 2nd and 3rd. ► APNKO mice have greater number of tumors, tumor area, inflammation and infiltration. ► APNKO treated mice showed significantly higher expression of pSTAT3 and Cox-2. ► APNKO treated mice showed significantly reduced expression of pAMPK. ► Higher expression of Cox-2 is present in the tumor area of APNKO mice than WT mice.

Keywords: Adiponectin; Inflammation; Colon; Cancer

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart by Mauricio Ibacache; Gina Sanchez; Zully Pedrozo; Felipe Galvez; Claudio Humeres; Ghislaine Echevarria; Juan Duaso; Mario Hassi; Lorena Garcia; Diaz-Araya Guillermo Díaz-Araya; Sergio Lavandero (pp. 537-545).

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30min of regional ischemia followed by 120min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning ( in vivo model) and peri-insult protection ( ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.► Dexmedetomidine activates Erk 1/2, Akt and eNOS in rat cardiac tissue. ► Dexmedetomidine reduces myocardial infarction size in models of regional I/R injury. ► Dexmedetomidine improves ventricular function recovery in a model of I/R injury. ► All these effects are mediated by cardiomycyte α₂-adrenergic receptor stimulation.

Keywords: Abbreviations; AAR; area at risk; ADCA; anterior descendent coronary artery; Akt; protein kinase B; CPP; coronary perfusion pressure; DEX; dexmedetomidine; EGF; epidermal growth factor; Erk1/2; extracellular signal-regulated kinase 1/2; eNOS; endothelial nitric oxide synthase; HR; heart rate; I/R; ischemia and reperfusion; LY; LY 294002; LV; left ventricle; LVDP; left ventricle develop pressure; LVEDP; left ventricle end-diastolic pressure; LVSP; left ventricle systolic pressure; MAP; mean arterial pressure; PI3K; phosphatidylinositol 3-kinase; Ser; serine; Thr; threonine; Tyr; tyrosine; YOH; yohimbine; +; dP/dt; _max; maximum rate of pressure change in the ventricle; −; dP/dt; _min; minimum rate of pressure change in the ventricleMyocardial ischemia; Myocardial infarction; Alpha2-adrenergic receptor; Dexmedetomidine; Anesthesia

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease by Olivier Thibault; Tristano Pancani; Philip W. Landfield; Christopher M. Norris (pp. 546-549).

Increased function of neuronal L-type voltage-sensitive Ca2⁺ channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal “zipper” slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca2⁺ dysregulation can differ substantially between animal models of normal aging and models of pathological aging.► Increased activity of L-type Ca2⁺ channels is linked to cognitive decline with aging. ► L channel activity has not been assessed in mouse models of Alzheimer's disease (AD). ► L channel activity was investigated in hippocampal “zipper” slices from APP/PS1 mice. ► Surprisingly, APP/PS1 mice showed reduced L channel activity compared to wild types. ► Ca2⁺ dysregulation may be independent of L channels in some mouse models of AD.

Keywords: Calcium dyshomeostasis; Alzheimer's disease; Hippocampus; Cognitive impairment

Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells by Robert Pazdro; John R. Burgess (pp. 550-556).

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.►Vitamin E protects neuronal cells against macromolecule damage induced by AGEs. ►Vitamin E has no effect on maintenance of intracellular reduced-state glutathione. ►Thiol NAC prevents AGE-induced neurite degeneration, but vitamin E has no effect. ►Thiol maintenance is a mechanism most associated with protection of neurites.

Keywords: Abbreviations; NAC; N-acetyl cysteine; AGEs; advanced glycation end products; MDA; malondialdehyde; PUFA; polyunsaturated fatty acids; RA; retinoic acid; α-T; α-tocopherolAdvanced glycation end product; Diabetes; Oxidative stress; Vitamin E; Neuropathy; N-acetyl cysteine

St. John's Wort inhibits insulin signaling in murine and human adipocytes by Allison J. Richard; Zhaleh J. Amini; David M. Ribnicky; Jacqueline M. Stephens (pp. 557-563).

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.► St. John's Wort (SJW) induces insulin resistance in 3T3-L1 and human adipocytes. ► SJW impairs IRS-1 tyrosine activation in murine and human fat cells. ► Hypericin and hyperforin are considered the primary active ingredients of SJW. ► Hypericin and hyperforin do not mediate effects of SJW in 3T3-L1 or human fat cells. ► SJW may contribute to or exacerbate adipocyte related diseases.

Keywords: Abbreviations; SJW; St. John's Wort; HI; hypericin; HF; hyperforin; T2DM; Type 2 diabetes mellitus; IRS-1; insulin receptor substrate 1; IRS-1 pY; ⁸⁹⁶; IRS-1 phosphorylated at tyrosine 896Fat cell; Insulin action; Botanicals; Hypericin; Hyperforin

Photosensitized reactions mediated by the major chromophore arising from glucose decomposition, result in oxidation and cross-linking of lens proteins and activation of the proteasome by Avila Felipe Ávila; Sebastian Trejo; Martin A. Baraibar; Bertrand Friguet; Eduardo Silva (pp. 564-572).

Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D₂O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.► Glucose degradation generates a chromophore with photosensitizing properties. ► Like in cataract photochemical protein crosslinking and oxidation were observed. ► Simultaneously, the chymotrypsin-like activity of the eye lens proteasome is activated.

Keywords: Cataract; Glucose degradation products; Photosensitization; Protein crosslinking; Proteasome; Aging

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint by Yvan Chen; Shang-Ting Fang; Pei-Chi Yeh; Hsueh-Hui Yang; Shin-Yuan Chen; Chih-Jui Chang; Wei-Jun Zhai; Yi Cheng Chen; Yue-Li Juang (pp. 573-580).

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.► PARK2 localizes to centrosome and its protein levels do not fluctuate through the cell cycle. ► Ectopic expression of PARK2 C-terminus truncation impairs the mitotic checkpoint and causes cell death. ► Ectopic expression of PARK2ΔC40 causes an accumulation of multinucleated cells. ► PARK2ΔC40 loses the ability of self-interaction, and has reduced solubility and stability.

Keywords: PARK2; Mitosis; Centrosome; Spindle assembly checkpoint; Self-interaction

Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C²⁰¹-Y or C⁴⁸⁹-S substitution associated with severe hypophosphatasia by Yasuhito Satou; Hiba A. Al-Shawafi; Sara Sultana; Saori Makita; Miwa Sohda; Kimimitsu Oda (pp. 581-588).

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171–172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-β- N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.► Tissue-nonspecific alkaline phosphatase mutants associated with hypophosphatasia. ► Lack of an intra-subunit disulfide-bond. ► Misfolding and degradation in proteasome. ► Diminished cell surface appearance explains molecular basis of the disease.

Keywords: Abbreviations; ALP; alkaline phosphatase; DMEM; Dulbecco's modified minimum essential medium; ER; endoplasmic reticulum; GPI; glycosylphosphatidylinositol; HPP; hypophosphatasia; LLnL; N; -acetyl-L-leucinyl-L-leucinyl-L-norleucinal; PI-PLC; phosphatidylinositol-specific phospholipase C; PLAP; placental alkaline phosphatase; TNSALP; tissue-nonspecific alkaline phosphatase; TNSALP (C201Y); TNSALP with a cysteine to tyrosine substitution at position 201; TNSALP (C489S); TNSALP with a cysteine to serine substitution at position 489; TNSALP (W); the wild-type TNSALPDegradation; Disulfide bond; Hypophosphatasia; Proteasome; Tissue-nonspecific alkaline phosphatase; Ubiquitination

Next-generation sequencing identifies TGF-β1-associated gene expression profiles in renal epithelial cells reiterated in human diabetic nephropathy by Eoin P. Brennan; Melissa J. Morine; David W. Walsh; Sarah A. Roxburgh; Maja T. Lindenmeyer; Derek P. Brazil; Peadar Ó. Gaora; Helen M. Roche; Denise M. Sadlier; Clemens D. Cohen; Catherine Godson; Finian Martin (pp. 589-599).

Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI — encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker — E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.► TGF-β1 is implicated in renal tubule injury in diabetic nephropathy (DN). ► RNA-Seq identifies a TGF-β1-driven signal in renal tubule epithelial cells. ► ARK5 is upregulated by TGF-β1 and necessary for complete TGF-β1 signaling. ► Defining TGF-β1signaling in renal epithelials will aid our understanding of DN.

Keywords: Diabetic nephropathy; Renal tubule; Epithelial–mesenchymal transition; Transforming growth-factor-beta1

S100 proteins in cartilage: Role in arthritis by Raghunatha R. Yammani (pp. 600-606).

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.► S100 family of protein in cartilage. ► Biological functions of S100 proteins in the cartilage. ► Role of S100 proteins in the development of arthritis.

Keywords: S100 protein; Cartilage; Chondrocyte; Arthritis; Osteoarthritis (OA)

Non-histone lysine acetylated proteins in heart failure by Jean Michel Grillon; Keven R. Johnson; Kumar Kotlo; Robert S. Danziger (pp. 607-614).

Both histone-acetylations and histone deacetylases have been shown to play a key role in cardiac remodeling. Recently, it has become abundantly clear that many non-histone proteins are modified by post-translational lysine acetylations and that these acetylations regulate protein activity, conformation, and binding. In the present study, non-histone acetylated proteins associated with heart failure were identified. Global screening for lysine acetylated proteins was performed using 2-dimensional gel electrophoresis coupled with immunoblotting with a primary monoclonal anti-acetyl-lysine antibody. Lysine acetylated proteins were compared in two rodent models of hypertensive heart failure, the Dahl salt-sensitive (SS) and spontaneously hypertensive heart failure prone (SHHF) rats with those in corresponding controls, i.e., the Dahl salt-resistant (SR) and W (W) rat strains, respectively. Forty-one and 66 acetylated proteins were detected in SS and SHHF failing hearts, respectively, but either not detected or detected with less abundance in corresponding control hearts. Twelve of these acetylated proteins were common to both models of heart failure. These were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry followed by Mascot Analysis and included mitochondrial enzymes: ATP synthase, long-chain acyl-CoA dehydrogenase, creatine kinase, malate dehydrogenase, and pyruvate dehydrogenase. The abundance of NAD-dependent deacetylase sirtuin-3 (Sirt3), a mitochondrial deacetylase was reduced in SS and SHHF failing hearts. This is the first description of non-histone protein acetylations associated with heart failure and raises the prospect that acetylations of mitochondrial proteins linked to reduced Sirt3 mediate, in part, metabolic changes in heart failure.► Non-histone acetylated proteins associated with heart failure were identified. ► Dahl salt-sensitive and spontaneously hypertensive heart failure rats were used. ► Global screening was done using 2D gel electrophoresis coupled to immunoblotting. ► Twelve lysine acetylated proteins were common to both models of heart failure. ► Mitochondrial Sirt3 was reduced in failing hearts.

Keywords: Abbreviations; C.I.; confidence interval; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HATs; histone acetyltransferases; HDACs; histone deacetylases; HDACis; histone deacetylase inhibitors; LCAD; long-chain acyl-CoA dehydrogenase; LV EF; left ventricular ejection fraction; LV PWT; left ventricular posterior wall thickness; MDH; malate dehydrogenase; SHHF; spontaneously hypertensive heart failure prone; Sirt3; Sirtuin-3; SR; Dahl salt-resistant; SS; Dahl salt-sensitive; W; WHeart failure; Lysine acetylation; Sirtuin-3; Mitochondrial protein; Global screening

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BBA - Molecular Basis of Disease (v.1822, #4)