Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.     

Risk of second bone sarcoma following childhood cancer: role of radiation therapy treatment

Bone sarcoma as a second malignancy is rare but highly fatal. The present knowledge about radiation-absorbed organ dose–response is insufficient to predict the risks induced by radiation therapy techniques. The objective of the present study was to assess the treatment-induced risk for bone sarcoma following a childhood cancer and particularly the related risk of radiotherapy. Therefore, a retrospective cohort of 4,171 survivors of a solid childhood cancer treated between 1942 and 1986 in France and Britain has been followed prospectively. We collected detailed information on treatments received during childhood cancer. Additionally, an innovative methodology has been developed to evaluate the dose–response relationship between bone sarcoma and radiation dose throughout this cohort. The median follow-up was 26 years, and 39 patients had developed bone sarcoma. It was found that the overall incidence was 45-fold higher [standardized incidence ratio 44.8, 95 % confidence interval (CI) 31.0–59.8] than expected from the general population, and the absolute excess risk was 35.1 per 100,000 person-years (95 % CI 24.0–47.1). The risk of bone sarcoma increased slowly up to a cumulative radiation organ absorbed dose of 15 Gy [hazard ratio (HR) = 8.2, 95 % CI 1.6–42.9] and then strongly increased for higher radiation doses (HR for 30 Gy or more 117.9, 95 % CI 36.5–380.6), compared with patients not treated with radiotherapy. A linear model with an excess relative risk per Gy of 1.77 (95 % CI 0.6213–5.935) provided a close fit to the data. These findings have important therapeutic implications: Lowering the radiation dose to the bones should reduce the incidence of secondary bone sarcomas. Other therapeutic solutions should be preferred to radiotherapy in bone sarcoma-sensitive areas.     

Synergistic Therapeutic Effect of Cisplatin and Phosphatidylinositol 3-Kinase (PI3K) Inhibitors in Cancer Growth and Metastasis of Brca1 Mutant Tumors

Drug resistance and cancer metastasis are two major problems in cancer research. During a course of therapeutic treatment in Brca1-associated tumors, we found that breast cancer stem cells (CSCs) exhibit an intrinsic ability to metastasize and acquire drug resistance through distinct signaling pathways. Microarray analysis indicated that the cytoskeletal remodeling pathway was differentially regulated in CSCs, and this was further evidenced by the inhibitory role of reagents that impair this pathway in the motility of cancer cells. We showed that cisplatin treatment, although initially inhibiting cancer growth, preventing metastasis through blocking cytoskeletal remodeling, and retarding CSC motility, eventually led to drug resistance associated with a marked increase in the number of CSCs. This event was at least partially attributed to the activation of PI3K signaling, and it could be significantly inhibited by co-treatment with rapamycin. These results provide strong evidence that cytoskeletal rearrangement and PI3K/AKT signaling play distinct roles in mediating CSC mobility and viability, respectively, and blocking both pathways synergistically may inhibit primary and metastatic cancer growth.     

TREC-IN: gene knock-in genetic tool for genomes cloned in yeast

Background

With the development of several new technologies using synthetic biology, it is possible to engineer genetically intractable organisms including Mycoplasma mycoides subspecies capri (Mmc), by cloning the intact bacterial genome in yeast, using the host yeast’s genetic tools to modify the cloned genome, and subsequently transplanting the modified genome into a recipient cell to obtain mutant cells encoded by the modified genome. The recently described tandem repeat coupled with endonuclease cleavage (TREC) method has been successfully used to generate seamless deletions and point mutations in the mycoplasma genome using the yeast DNA repair machinery. But, attempts to knock-in genes in some cases have encountered a high background of transformation due to maintenance of unwanted circularization of the transforming DNA, which contains possible autonomously replicating sequence (ARS) activity. To overcome this issue, we incorporated a split marker system into the TREC method, enabling seamless gene knock-in with high efficiency. The modified method is called TREC-assisted gene knock-in (TREC-IN). Since a gene to be knocked-in is delivered by a truncated non-functional marker, the background caused by an incomplete integration is essentially eliminated.

Results

In this paper, we demonstrate applications of the TREC-IN method in gene complementation and genome minimization studies in Mmc. In the first example, the Mmc dnaA gene was seamlessly replaced by an orthologous gene, which shares a high degree of identity at the nucleotide level with the original Mmc gene, with high efficiency and low background. In the minimization example, we replaced an essential gene back into the genome that was present in the middle of a cluster of non-essential genes, while deleting the non-essential gene cluster, again with low backgrounds of transformation and high efficiency.

Conclusion

Although we have demonstrated the feasibility of TREC-IN in gene complementation and genome minimization studies in Mmc, the applicability of TREC-IN ranges widely. This method proves to be a valuable genetic tool that can be extended for genomic engineering in other genetically intractable organisms, where it may be implemented in elucidating specific metabolic pathways and in rationale vaccine design.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1180) contains supplementary material, which is available to authorized users.     

Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.     

Control of Promatrilysin (MMP7) Activation and Substrate-specific Activity by Sulfated Glycosaminoglycans

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-α-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8–12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12–24 h for 50% conversion. These inefficient reactions suggest that natural cofactors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (>50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (K_D, 400 nm) and CS-E (K_D, 630 nm). Active matrilysin bound heparin (K_D, 150 nm) but less so to CS-E (K_D, 60 μm). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.Matrix metalloproteinases (MMPs)³ comprise a family of endopeptidases that act on a variety of extracellular proteins, such as chemokines, antimicrobial peptides, matrix components, and more, to effect numerous repair, immune, and disease processes (1–3). For many substrates, MMP cleavage results in gain-of-function processing, such as the activation of latent antimicrobial peptides (4, 5) and cytokines (1), or altered biologic activity, as with limited proteolysis of chemokines (6, 7) and shedding of cell surface proteins (8). Thus, the mechanisms controlling zymogen activation and proteinase activity against specific substrates would sit high in the hierarchy of events controlling many host response pathways. As for all proteinases, the activity of MMPs is regulated at four points: gene expression, compartmentalization (i.e. pericellular accumulation of enzyme), proenzyme (or zymogen) activation, and enzyme inactivation, and is further controlled by substrate availability, concentration, and affinity.ProMMPs are kept in a catalytically inactive state by the interaction between the thiol of the conserved prodomain cysteine and the zinc ion of the catalytic site. To become active, the thiol-Zn²⁺ interaction, commonly called the “cysteine switch,” must be disrupted (9), which can be mediated by proteolysis of the prodomain, post-translational modification of the thiol, allosteric interactions with other macromolecules, or other possible mechanisms (10). About one-third of proMMPs contains a furin-recognition sequence and are activated in the secretion pathway by furin proprotein convertase cleavage of the prodomain. However, with the possible exception of proMMP2 activation by MMP14, the physiologic activation mechanism of most MMPs is not known (10).Matrilysin (28 kDa zymogen, 19 kDa active enzyme) is expressed by mucosal epithelia and some macrophages and functions as a key effector of repair and immunity. Established functions of matrilysin include facilitating re-epithelialization (11, 12), cleaving Fas ligand to promote apoptosis (13, 14), shedding syndecan-1 to control neutrophil influx (15), and macrophage-mediated elastolysis (16).In mice, matrilysin activates pro-α-defensins (procryptdins), a family of structurally similar 3–4 kDa antimicrobial peptides found in the granules of Paneth cells at the base of the crypts of Lieberkühn (17). Because of the lack of mature cryptdins, matrilysin-null (Mmp7^−/−) mice have an impaired ability to battle enteric pathogens (4). Cryptdins are packaged as pro-proteins of 7–8 kDa and are cleaved at a conserved site by matrilysin within the secretion granules (4, 18). In resting Paneth cells, the steady-state levels of pro- and activated cryptdins are roughly equivalent. Upon stimulation, the balance of procryptdins is rapidly activated indicating efficient proteolysis by matrilysin within the secretory pathway (18, 19). However, in defined in vitro reactions containing just substrate and proteinase, activation of procryptdins by matrilysin is slow, with only 50% of the precursor cleaved in 8 h or longer (4). Furthermore, both pro- and active matrilysin are present in Paneth cells granules (4) indicating that this MMP is activated in vivo by prodomain cleavage. The inefficient cleavage of procryptdins in vitro, their rapid processing in vivo, and the presence of activated matrilysin in Paneth cell granules led us to hypothesize that other factors regulate both the activation of promatrilysin and its activity against physiologic substrates.Yu et al. (20, 21) reported that heparin increases matrilysin activity about 2–4-fold in a transferrin zymogram assay, and they reported that matrilysin colocalizes to heparan sulfate molecules in tissue. However, transferrin is not a physiologic substrate of this MMP, and it is not known how heparin and other glycosaminoglycans affect matrilysin activity against established substrates, such as procryptdins. Therefore, we assessed matrilysin activity in vitro in the presence of various glycosaminoglycans (GAGs), and we found that both zymogen activation and activity against specific substrates are markedly enhanced by highly sulfated molecules. Our findings suggest that specific GAGs function to control matrilysin proteolysis.