51 research outputs found
Butyl Rubber-Aliphatic Polyester Graft Copolymers for Biomedical Applications: Synthesis and Analysis of Chemical, Physical and Biological Properties
Biomaterials can be used in a wide variety of medical applications owing to their breadth of characteristics that can be imparted by varying their chemical structures. Butyl rubber (IIR), which is a copolymer of isobutylene (IB) and small percentages of isoprene (IP), is particularly attractive as a biomaterial because of its elastomeric mechanical properties, biocompatibility, impermeability and high damping characteristics. IIR is typically vulcanized through chemical-based crosslinking mechanisms. However, these methods are not acceptable for biological applications. This thesis focuses on the synthesis of IIR-polyester graft copolymers by grafting biodegradable and biocompatible polyesters including poly(caprolactone) (PCL) and poly(d,l-lactide) (PDLLA) to the IIR backbone, and on the study of their properties
Investigating the interplay between protein kinases and caspases
The balance between cell survival and death is a crucial process in human development and tissue homeostasis, but is also misregulated in disease. In large part, apoptosis is controlled by caspases, a hierarchical series of cysteine aspartic acid proteases that demolish the cell by cleaving key structural and enzymatic proteins, but emerging paradigms have highlighted the ability of kinases to regulate caspase activity. One way in which kinases can control the progression of apoptosis is through phosphorylation of caspase substrates, which acts to prevent caspase cleavage of that target.
In this thesis, we develop new strategies to study this regulatory mechanism, and focus particularly on protein kinase CK2; a kinase with enigmatic regulation, an obvious, but undefined role in anti-apoptotic signaling, and demonstrated value as a candidate for targeted therapy of cancer. First, we used predictive strategies for identifying CK2 substrates that, when phosphorylated, regulate proteolysis by caspases. Interestingly, we predicted and validated that phosphorylation of caspase-3 itself by CK2 prevents its cleavage and activation, representing a putative, novel mechanism by which CK2 might control apoptosis in disease. We then utilized cell models that over-expressed different forms of CK2, and found that caspase-3 phosphorylation was specific only for the CK2a¢ subunit in the absence of CK2b - a level of isozyme specificity not before seen for CK2. Interestingly, documented misregulation of this isoform in disease suggests that the phenotypes generated by varied CK2 levels in cancer may well depend on the form of CK2 present. We also establish the utility of proteomic applications for identifying novel caspase substrates whose cleavage is regulated by phosphorylation. Interestingly, we noted that phosphorylation can also promote caspase substrate cleavage, likely through a mechanism in which phosphorylation alters scissile bond accessibility.
Collectively, our work provides insight into the regulation of protein kinase CK2 and demonstrates that the specialized functions of different forms may define cellular phenotypes in disease. Furthermore, we identified a number of caspase substrates whose cleavage is regulated by phosphorylation, thereby validating our novel approaches and delineating putative apoptotic control points
Synthesis, properties, and degradation of polyisobutylene-polyester graft copolymers
The development of copolymers is a promising approach for combining the favorable properties of two polymers and obtaining new properties of the combination. In this work, graft copolymers of polyisobutylene (PIB) and polycaprolactone (PCL) or poly(D,L-lactide) (PDLLA) were synthesized and studied. Amine terminated polyesters were synthesized and were grafted onto an activated PIB backbone synthesized from butyl rubber, a copolymer of isobutylene and 2 mol % isoprene. The polyester content was tuned from 15 to 44 wt % by varying the molar mass of the polyester blocks and the number of molar equivalents used in the grafting reaction. The graft copolymers with higher polyester content underwent nanoscale phase separation, as demonstrated by differential scanning calorimetry and atomic force microscopy imaging. This was found to provide enhanced mechanical properties such as increased tensile strength and Young’s modulus relative to the starting rubber or physical blends. Despite the significant polyester content of the graft copolymers and the susceptibility of the polyesters to degradation, the graft copolymers underwent negligible mass loss in 5 M NaOH over a period of 8 weeks. These results suggest that polyesters can be incorporated into PIB to tune and enhance its properties, while maintaining high chemical stability
An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation
Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc
An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation
Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis
Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification of bona fide Protein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2
bS Supporting Informatio
Versatile Strategy for Biochemical, Electrochemical and Immunoarray Detection of Protein Phosphorylations
Protein kinases catalyze the phosphorylation of cellular
proteins
involved in the regulation of many cellular processes and have emerged
as promising targets for the treatment of several diseases. Conventional
assays to monitor protein kinase activity are limited because they
typically rely on transfer of radioactive phosphate or phospho-specific
antibodies that recognize specific substrates or sequence motifs.
To overcome the limitations of conventional assays, we have developed
a versatile approach based on transfer of ferrocene-phosphate that
can be readily monitored using electrochemical detection or detection
with antiferrocene antibodies in an immunoarray format. This assay
is readily adapted to multiplex arrays and can be employed for monitoring
kinase activity in complex mixtures and for kinase inhibitor profiling
Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3
Covalent Polyisobutylene-Paclitaxel Conjugates for Controlled Release from Potential Vascular Stent Coatings
© 2015 American Chemical Society. The development of covalent polyisobutylene (PIB)-paclitaxel (PTX) conjugates as a potential approach to controlling drug release from vascular stent coatings is described. PIB-PTX materials containing ∼24 and ∼48 wt % PTX, conjugated via ester linkages, were prepared. The PTX release profiles were compared with those of physical mixtures of PTX with carboxylic acid-functionalized PIB and with the triblock copolymer polystyrene-b-PIB-b-polystyrene (SIBS). Covalent conjugation led to significantly slower drug release. Atomic force microscopy imaging of coatings of the materials suggested that the physical mixtures exhibited multiple domains corresponding to phase separation, whereas the materials in which PTX was covalently conjugated appeared homogeneous. Coatings of the conjugated materials on stainless steel surfaces suffered less surface erosion than the physically mixed materials, remained intact, and adhered well to the surface throughout the thirty-five day study. Tensile testing and rheological studies suggested that the incorporation of PTX into the polymer introduces similar physical changes to the PIB as the incorporation of a glassy polystyrene block does in SIBS. Cytotoxicity assays showed that the coatings did not release toxic levels of PTX or other species into a cell culture medium over a 24 h period, yet the levels of PTX in the materials were sufficient to prevent C2C12 cells from adhering to and proliferating on them. Overall, these results indicate that covalent PIB-PTX conjugates have promise as coatings for vascular stents
Mechanical analysis and characterization of IGUs with different silicone sealed spacer connections - Part 1: experiments
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