Artificial DnaJ Protein for protein production and conformational diseases.

For secreted proteins, proper protein folding is essential not only for biological function but also for secretion itself. Proteins with folding problems are trapped in the endoplasmic reticulum (ER) and are eventually degraded in the cytoplasm. In this study, we exploited co-expression of an artificial fusion protein, based on the sequence of a DnaJ protein, which could interact as co-chaperones in the Hsp70-based protein-folding system, with target recombinant secreted proteins to enhance their production and secretion. The J-domain sequence or a fragment thereof was conjugated to a target protein-binding domain that was capable of binding to a portion of the target-protein sequence. Production of many of the target proteins was significantly upregulated when co-expressed with the J-domain fusion protein. Surprisingly, the enhancement of secretion was observed even when the J-domain had a mutation in the HPD motif, which is necessary for J-protein-Hsp70 interactions, suggesting the phenomenon observed is independent on functional J-protein-Hsp70 interactions. This technology has great potential for not only enhancing the production of recombinant proteins, but also to treat conformational diseases such as cystic fibrosis, and Alpha-1 antitrypsin deficiency.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling.

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype in vivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity.

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.

BAG3 (BCL2-associated athanogene 3) interacts with MMP-2 to positively regulate invasion by ovarian carcinoma cells.

BAG3 (BCL2-associated athanogene 3) is a member of the BAG family proteins, which interact with and regulate Hsp70. Our aim in the present study was to correlate BAG3 expression in ovarian cancer with invasion and progression-free survival. We found that BAG3 interacts with MMP2 in cultured ovarian cancer cells, and that knockdown of BAG3 expression downregulated MMP2 expression and diminished cell motility and invasiveness. The incidence of BAG3 positivity was significantly higher at advanced clinical stages of ovarian cancer than at early stages. We suggest that BAG3 binds to MMP2 to positively regulate the process of cell invasion.

BAG3 and Hsc70 interact with actin capping protein CapZ to maintain myofibrillar integrity under mechanical stress.

RATIONALE: A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood. OBJECTIVE: bag3(-/-) mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress. METHODS AND RESULTS: To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and a short hairpin RNA-mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZß1, by promoting association between Hsc70 and CapZß1. BAG3 facilitates the distribution of CapZß1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin-proteasome-mediated degradation. Inhibition of CapZß1 function by overexpressing CapZß2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZß1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3(-/-) mice exhibited myofibrillar degeneration and lost contractile activity after caffeine contraction. CONCLUSIONS: These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion.

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.

BAG3 regulates motility and adhesion of epithelial cancer cells.

BAG3 protein binds to and regulates Hsp70 chaperone activity. The BAG3 protein contains a WW domain and a proline-rich region with SH3-binding motifs, suggesting that it may interact with proteins relevant to signal transduction, recruiting Hsp70 to signaling complexes and altering cell responses. BAG3 overexpression has been observed in human cancers. We show here that homozygous BAG3-deficient mouse embryonic fibroblasts (MEF) exhibit delayed formation of filopodia and focal adhesion complexes when freshly plated. BAG3-deficient MEFs show reduced cell motility in culture. We observed that endogenous BAG3 protein is highly expressed in many human epithelial cancer cell lines, especially adenocarcinomas. Gene transfer-mediated overexpression of BAG3 increased motility of Cos7 cell and several human cancer cell lines, including breast cancer MCF7 and prostate cancer DU145 and ALVA31 cell lines. Conversely, reduction of BAG3 protein by RNA interference (RNAi) decreased cell motility in four of four epithelial tumor lines tested. We observed an influence of BAG3 on cell adhesion in culture. In Cos7 kidney epithelial cells, BAG3 protein partially colocalizes with actin at the leading edge of migrating cells, wherein active actin polymerization and nucleation occur. RNAi-mediated reductions in BAG3 expression were associated with decreased Rac1 activity, suggesting a role for BAG3 in regulating this small GTPase involved in actin-cytoskeleton dynamics. In mice, RNAi-mediated reductions in BAG3 in a human tumor xenograft suppressed invasion and metastasis in vivo. Thus, the high levels of BAG3 protein seen in some epithelial cancer cell lines may be relevant to mechanisms of tumor invasion and metastasis.

A novel ubiquitin-binding protein ZNF216 functioning in muscle atrophy.

The ubiquitin-proteasome system (UPS) is critical for specific degradation of cellular proteins and plays a pivotal role on protein breakdown in muscle atrophy. Here, we show that ZNF216 directly binds polyubiquitin chains through its N-terminal A20-type zinc-finger domain and associates with the 26S proteasome. ZNF216 was colocalized with the aggresome, which contains ubiquitinylated proteins and other UPS components. Expression of Znf216 was increased in both denervation- and fasting-induced muscle atrophy and upregulated by expression of constitutively active FOXO, a master regulator of muscle atrophy. Mice deficient in Znf216 exhibited resistance to denervation-induced atrophy, and ubiquitinylated proteins markedly accumulated in neurectomized muscle compared to wild-type mice. These data suggest that ZNF216 functions in protein degradation via the UPS and plays a crucial role in muscle atrophy.

A RANKL-inducible gene Znf216 in osteoclast differentiation.

Osteoclasts possess catabolic activity in mineralized tissues and are involved in bone remodeling coordinating with osteoblasts. Although the pathway using receptor and activator of NF-kappa B (RANK) and its ligand, RANKL, is known to be essential for osteoclast differentiation, their precise mechanisms are not fully understood. Using DNA microarray technology, we searched for genes that were up-regulated after RANKL stimulation in the macrophage cell line, RAW264.7 cells. A gene, Znf216, which encodes a zinc-finger protein, was detected among those genes up-regulated after RANKL stimulation. Expression of Znf216 was also induced by other cytokines such as TNFalpha and IL-1beta. Although ectopic expression of full-length ZNF216 abrogated osteoclast differentiation, its truncated forms accelerated it. No significant inhibitory effect on the NF-kappa B pathway was observed, however. These results suggest that ZNF216 is a potent inhibitory factor for osteoclast differentiation and that the mechanism is unlikely due to direct attenuation of the NF-kappa B pathway.

Ataxia telangiectasia mutated (Atm) knockout mice as a model of osteopenia due to impaired bone formation.

ATM is a member of the PI-3 kinase protein family, encoded by the gene, ATM, responsible for ataxia telangiectasia (AT). AT is recognized as a genomic instability syndrome, sharing accelerated senescence symptoms in human and mouse. Here, we present evidence that the bone phenotype of Atm knockout (AtmKO) mice is similar to that observed in disuse and/or aging syndromes. A significant decrease in 3-dimensional bone volume fraction (BV/TV) of the fifth lumbar vertebra was observed in AtmKO mice by microCT, compared with heterozygous control mice at 10 weeks of age. Bone histomorphometry revealed that both BFR/BS and Oc.S/BS were significantly decreased in KO mice. To determine the cellular basis of this bone phenotype, we employed in vitro osteoclastogenesis and colony formation assays using bone marrow cells derived from KO and control mice. There was no difference in osteoclast formation in ex vivo cultures. CFU-F was markedly reduced in AtmKO-derived cultures compared with control mice, whereas differentiation of calvaria-derived osteoblasts did not differ between the genotypes. Furthermore, expression levels of IGF1R were significantly decreased, and p38 was aberrantly phosphorylated in marrow stromal cells from AtmKO mice. These results indicate that the pathogenesis of the osteopenic phenotype in AtmKO mice is similar to that of disuse and/or aging syndromes and is caused, at least in part, by a stem cell defect due to lack of IGF signaling.

Mouse models of senile osteoporosis.

Little is known about the pathophysiology of normal human and mouse senescence. On the other hand, the pathology of age-related disorders, such as senile osteoporosis, has been investigated. In vivo studies on the pathology of osteoporosis have been conducted primarily in rodents. Although mouse models of senile osteoporosis display some discrepancies relative to their human counterparts with regard to symptoms and pathology, these experimental models are useful and powerful tools for basic and preclinical studies. Here, we review existing mouse models of senile osteoporosis, including those exhibiting premature aging phenotypes, and discuss their pathogenesis, particularly with regard to age-related changes in stem cells.

Progeroid syndrome as a model for impaired bone formation in senile osteoporosis.

Senile or age-related/dependent osteoporosis is caused by reduced bone formation, rather than increased bone resorption as in postmenopausal osteoporosis. Here we review genetically engineered mouse models with defects in osteoblastic proliferation or differentiation with focus on IGF signaling and stem cells. Model mice for human progeroid syndromes may provide useful tools for studying the pathogenesis of senile osteoporosis.

Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow.

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1alpha,25-dihydroxyvitamin D3 [1a,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1alpha,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.