Multi-Tasking Role of the Mechanosensing Protein Ankrd2 in the Signaling Network of Striated Muscle

Background Ankrd2 (also known as Arpp) together with Ankrd1/CARP and DARP are members of the MARP mechanosensing proteins that form a complex with titin (N2A)/calpain 3 protease/myopalladin. In muscle, Ankrd2 is located in the I-band of the sarcomere and moves to the nucleus of adjacent myofibers on muscle injury. In myoblasts it is predominantly in the nucleus and on differentiation shifts from the nucleus to the cytoplasm. In agreement with its role as a sensor it interacts both with sarcomeric proteins and transcription factors. Methodology/Principal Findings Expression profiling of endogenous Ankrd2 silenced in human myotubes was undertaken to elucidate its role as an intermediary in cell signaling pathways. Silencing Ankrd2 expression altered the expression of genes involved in both intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-\u3b2 and Wnt signaling). The significance of Ankrd2 in cell signaling was strengthened by the fact that we were able to show for the first time that Nkx2.5 and p53 are upstream effectors of the Ankrd2 gene and that Ankrd1/CARP, another MARP member, can modulate the transcriptional ability of MyoD on the Ankrd2 promoter. Another novel finding was the interaction between Ankrd2 and proteins with PDZ and SH3 domains, further supporting its role in signaling. It is noteworthy that we demonstrated that transcription factors PAX6, LHX2, NFIL3 and MECP2, were able to bind both the Ankrd2 protein and its promoter indicating the presence of a regulatory feedback loop mechanism. Conclusions/Significance In conclusion we demonstrate that Ankrd2 is a potent regulator in muscle cells affecting a multitude of pathways and processes

The expression of Muscle ankyrin repeat proteins in brown adipose tissue

MARP family members CARP, Ankrd2 and DARP are expressed in the striated muscle, while DARP protein is also detected in brown adipose tissue (BAT). Taking into account recent findings concerning the common origin of muscle and brown fat, expression of CARP and Ankrd2 in mouse BAT was investigated. We demonstrated Ankrd2 expression in both inactive and thermogenically active BAT, while CARP expression was not detected. Our findings suggest that the expression of Ankrd2 in BAT could be a part of the 'myogenic transcriptional signature', further supporting the evidence that muscle and brown adipose cells arise from the same myoblastic precursor

AggLb Is the Largest Cell-Aggregation Factor from <i>Lactobacillus paracasei</i> Subsp. <i>paracasei</i> BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion <i>In Vitro</i>

<div><p>Eleven <i>Lactobacillus</i> strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of <i>Lactobacillus paracasei</i> subsp. <i>paracasei</i> BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the <i>aggLb</i> gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of <i>aggLb</i> causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the <i>aggLb</i> gene of BGNJ1-64 enabled detection of the same type of <i>aggLb</i> gene in five of eleven selected aggregation-positive <i>Lactobacillus</i> strains. Heterologous expression of <i>aggLb</i> confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.</p></div

Functional analysis of novel alpha-1 antitrypsin variants G320R and V321F

Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure. © 2014 Springer Science+Business Media Dordrecht

List of bacterial strains and plasmids used in the study.

<p>List of bacterial strains and plasmids used in the study.</p

Screening of selected Agg⁺ strains for presence of <i>aggLb</i> gene by PCR.

<p>Agarose gel electrophoresis of <i>aggLb</i> gene fragments obtained by PCR amplification with different primers (Agg1 Fw—Agg1 Rev; lanes a), (Agg2 Fw—Agg2 Rev; lanes b), (Agg3 Fw—Agg3 Rev; lanes c). Lanes: 1- GeneRuler 1 kb Plus DNA Ladder; 2- BGSJ2-8; 3- BGAR75; 4- BGGR2-68; 5- BGGR2-82; 6- BGDP1-84; 7- BGDP9-38; 8- BGNJ1-3; 9- BGNJ1-61; 10- BGNJ1-64; 11- BGNJ1-70; 12-BGZLS30-6.</p

Schematic representation of AggLb protein.

<p>Boxes indicate domains of protein. Arrows indicate repeats.</p

Microbial adhesion to solvents.

<p>The percent adhesion of selected strains to different solvents was determined by the MATS assay. The values are the mean of three independent experiments and error bars represent standard deviations.</p