The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Characterization of a microbial transglutaminase cross-linked type II collagen scaffold

This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of ε-(γ-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 ± 0.03 kPa) compared to controls (0.21 ± 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase cross-linked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications. © Mary Ann Liebert, Inc.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

CNK1 scaffold protein and its role in the regulations of NF-KB signalling

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The Great American Scaffold:intertextuality and identity in American presidential discourse

Based on extensive quantitative and qualitative analyses of a corpus of American presidential speeches that includes all inaugural addresses and State of the Union messages from 1789 to 2008, as well as major foreign and security policy speeches after 1945, this research monograph analyzes the various forms and functions of intertextual references found in the discourse of American presidents. Working within an original, interdisciplinary theoretical framework established by theories of intertextuality, discourse analysis, and presidential studies, the book discusses five different types of presidential intertextuality, all of which contribute jointly to creating a set of carefully manipulated and politically powerful images of both the American nation and the American presidency. The book is intended for scholars and students in political and presidential studies, communications, American cultural studies, and linguistics, as well as anyone interested in the American presidency in general.

Compartmentalization of the MAPK scaffold protein KSR1 modulates synaptic plasticity in hippocampal neurons

ERK1/2 is required for certain forms of synaptic plasticity, including the long-term potentiation of synaptic strength. However, the molecular mechanisms regulating synaptically localized ERK1/2 signaling are poorly understood. Here, we show that the MAPK scaffold protein kinase suppressor of Ras 1 (KSR1) is directly phosphorylated by the downstream kinase ERK1/2. Quantitative Western blot analysis further demonstrates that expression of mutated, feedback-deficient KSR1 promotes sustained ERK1/2 activation in HEK293 cells in response to EGF stimulation, compared to a more transient activation in control cells expressing wild-type KSR1. Immunocytochemistry and confocal imaging of primary hippocampal neurons from newborn C57BL6 mice further show that feedback phosphorylation of KSR1 significantly reduces its localization to dendritic spines. This effect can be reversed by tetrodotoxin (1 μM) or PD184352 (2 μM) treatment, further suggesting that neuronal activity and phosphorylation by ERK1/2 lead to KSR1 removal from the postsynaptic compartment. Consequently, electrophysiological recordings in hippocampal neurons expressing wild-type or feedback-deficient KSR1 demonstrate that KSR1 feedback phosphorylation restricts the potentiation of excitatory postsynaptic currents. Our findings, therefore, suggest that feedback phosphorylation of the scaffold protein KSR1 prevents excessive ERK1/2 signaling in the postsynaptic compartment and thus contributes to maintaining physiological levels of synaptic excitability. © FASEB.