Altered expression of beta-galactosidase-1-like protein 3 (Glb1l3) in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout mouse model of Leber's congenital amaurosis

Purpose: In this study, we investigated the expression of the gene encoding beta-galactosidase (Glb)-1-like protein 3 (Glb1l3), a member of the glycosyl hydrolase 35 family, during retinal degeneration in the retinal pigment epithelium (RPE)-specific 65-kDa protein knockout (Rpe65(-/-)) mouse model of Leber congenital amaurosis (LCA). Additionally, we assessed the expression of the other members of this protein family, including beta-galactosidase-1 (Glb1), beta-galactosidase-1-like (Glb1l), and beta-galactosidase-1-like protein 2 (Glb1l2).Methods: The structural features of Glb1l3 were assessed using bioinformatic tools. mRNA expression of Glb-related genes was investigated by oligonucleotide microarray, real-time PCR, and reverse transcription (RT) -PCR. The localized expression of Glb1l3 was assessed by combined in situ hybridization and immunohistochemistry.Results: Glb1l3 was the only Glb-related member strongly downregulated in Rpe65(-/-) retinas before the onset and during progression of the disease. Glb1l3 mRNA was only expressed in the retinal layers and the RPE/choroid. The other Glb-related genes were ubiquitously expressed in different ocular tissues, including the cornea and lens. In the healthy retina, expression of Glb1l3 was strongly induced during postnatal retinal development; age-related increased expression persisted during adulthood and aging.Conclusions: These data highlight early-onset downregulation of Glb1l3 in Rpe65-related disease. They further indicate that impaired expression of Glb1l3 is mostly due to the absence of the chromophore 11-cis retinal, suggesting that Rpe65 deficiency may have many metabolic consequences in the underlying neuroretina.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression.

Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal.

CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter.

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic beta-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and 280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic beta-cells where it functions as a transactivator of the GLUT2 gene.

Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle.

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3(-/-) mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3(-/-) mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3(-/-) muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3(-/-) skeletal muscle. The exaggerated ROS in the Smad3(-/-) muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy.

Patterns of calcium-binding proteins support parallel and hierarchical organization of human auditory areas.

The human primary auditory cortex (AI) is surrounded by several other auditory areas, which can be identified by cyto-, myelo- and chemoarchitectonic criteria. We report here on the pattern of calcium-binding protein immunoreactivity within these areas. The supratemporal regions of four normal human brains (eight hemispheres) were processed histologically, and serial sections were stained for parvalbumin, calretinin or calbindin. Each calcium-binding protein yielded a specific pattern of labelling, which differed between auditory areas. In AI, defined as area TC [see C. von Economo and L. Horn (1930) Z. Ges. Neurol. Psychiatr.,130, 678-757], parvalbumin labelling was dark in layer IV; several parvalbumin-positive multipolar neurons were distributed in layers III and IV. Calbindin yielded dark labelling in layers I-III and V; it revealed numerous multipolar and pyramidal neurons in layers II and III. Calretinin labelling was lighter than that of parvalbumin or calbindin in AI; calretinin-positive bipolar and bitufted neurons were present in supragranular layers. In non-primary auditory areas, the intensity of labelling tended to become progressively lighter while moving away from AI, with qualitative differences between the cytoarchitectonically defined areas. In analogy to non-human primates, our results suggest differences in intrinsic organization between auditory areas that are compatible with parallel and hierarchical processing of auditory information.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-kappaB activation.

Bcl10, a caspase recruitment domain (CARD)-containing protein identified from a breakpoint in mucosa-associated lymphoid tissue (MALT) B lymphomas, is essential for antigen-receptor-mediated nuclear factor kappaB (NF-kappaB) activation in lymphocytes. We have identified a novel CARD-containing protein and interaction partner of Bcl10, named Carma1. Carma1 is predominantly expressed in lymphocytes and represents a new member of the membrane-associated guanylate kinase family. Carma1 binds Bcl10 via its CARD motif and induces translocation of Bcl10 from the cytoplasm into perinuclear structures. Moreover, expression of Carma1 induces phosphorylation of Bcl10 and activation of the transcription factor NF-kappaB. We propose that Carma1 is a crucial component of a novel Bcl10-dependent signaling pathway in T-cells that leads to the activation of NF-kappaB.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.