Proteolysis of insulin-like growth factor binding proteins (IGFBPs) by calpain

Calpains are non-lysosomal, Ca2+-dependent cysteine proteases, which are ubiquitously distributed across cell types and vertebrate species. The rules that govern calpain specificity have not yet been determined. To elucidate the cleavage pattern of calpains, we carried out calpain-induced proteolytic studies on the insulin-like growth factor binding proteins IGFBP-4 and -5. Proteolysis of IGFBPs is well characterized in numerous reports. Our results show that calpain cleavage sites are in the non-conserved unstructured regions of the IGFBPs. Compilation of the calpain-induced proteolytic cleavage sites in several proteins reported in the literature, together with our present study, has not revealed clear preferences for amino acid sequences. We therefore conclude that calpains seem not to recognize amino acid sequences, but instead cleave with low sequence specificity at unstructured or solvent-exposed fragments that connect folded, stable domains of target proteins

Structure-property study of keto-ether polyimides

As part of an on-going effort to develop an understanding of how changes in the chemical structure affect polymer properties, an empirical study was performed on polyimides containing only ether and/or carbonyl connecting groups in the polymer backbone. During the past two decades the structure-property relationships in linear aromatic polyimides have been extensively investigated. More recently, work has been performed to study the effect of isomeric attachment of keto-ether polyimides on properties such as glass transition temperature and solubility. However, little work has been reported on the relation of polyimide structure to mechanical properties. The purpose of this study was to determine the effect of structural changes in the backbone of keto-ether polyimides on their mechanical properties, specifically, unoriented thin film tensile properties. This study was conducted in two stages. The purpose of the initial stage was to examine the physical and mechanical properties of a representative group (four) of polyimide systems to determine the optimum solvent and cure cycle requirements. These optimum conditions were then utilized in the second stage to prepare films of keto-ether polyimides which were evaluated for mechanical and physical properties. All of the polyimides were prepared using isomers of oxydianiline (ODA) and diaminobenzophenone (DABP) in combination with 3,3',4,4'-benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydiphthalic anhydride (ODPA)

Mechanisms and Integration of Signal Pathway: A Role for Calpains?

In order to survive cells must sense and respond to changes in their environment. Environmental cues trigger a variety of events within cells. The concentration and movements of calcium ions are essential regulators of many of these cellular responses. Proper control of intracellular calcium is essential because at thigh levels calcium can lead to cell damage or death. Calcium accomplishes it effects through binding to specific proteins such as calmodulin and calpain. Calmodulin, named for its ability to bind calcium and to modulate the activity of other cellular components, is an important mediator of calcium signals and its mechanism of action is relatively well understood. The calpains are proteolytic enzymes that are regulated by calcium binding. Proteolytic enzymes modify cellular proteins such that the target protein is destroyed or is altered in its function. Calpains can modify those proteins, called pumps or channels, that directly allow calcium ion movement and therefore calpains may be involved directly in helping cells to regulate calcium signals. Through its action on other key regulator proteins and enzymes, calpains might also help to integrate cellular responses that involve calcium with other events that do not. Although the activity of calpain is controlled by calcium its action may be prevented by another protein (calpastatin) that specifically inhibits these enzymes. Although calpastatin does not bind calcium directly it binds to calpain only when calpain has bound calcium. Thus the interactions between these two proteins is important in regulating calpain function. By comparison with calmodulin, there is little known about how calcium regulates calpain structure, function or its interactions with other proteins. Thus the immediate goals for this proposal are to understand more about the biochemical mechanisms that regulate calpain. This project aims to answer two major questions: 1) how does calcium binding to calpain regulate the enzymatic activity? 2) can a calpastatin insensitive calpain be generated by exploiting what is known about the interaction between calpain and calpastatin? An enzyme with this property will be useful, in future studies, for determining the role of calpains in cells. These goals will be accomplished through techniques of protein chemistry and expression of recombinant DNA molecules to generate modified enzymes or enzyme fragments for study. This knowledge will provide information on the role of calpain in critical cellular processes. %%% Calcium is important in the regulation of many essential intracellular processes. Many of the effecPts of calcium occur after it binds to specific proteins. One of these proteins is calpain. Calpains are proteolytic enzymes that are regulated by calcium binding, and in turn modify other cellular proteins in ways which often alter their function. Little is known of the mechanism by which calcium modifies calpain, and this project will study the biochemical mechanisms that regulate calpain and the role of calcium in these processes

The synthesis, characterization, and application of ether-containing polymides

Polyimides are a family of heterocyclic polymers that have received extensive evaluation as adhesives, fibers, films, moldings, composite matrices, insulators, coatings, membranes, and resists. their outstanding thermal stability, excellent mechanical and electrical properties, and chemical resistance make them attractive for many applications.;This research developed ether-containing polyimides for three applications: liquid crystalline polyimides as processing aids, polyimides for microelectronic applications, and polyimides for harsh environments. The approach consisted of three primary activities: (1) developed novel diether dianhydrides for polyimide fabrication, (2) designed, characterized, and evaluated polyimide architectures based on the material application requirements, and (3) provided extensive structure-property relationships utilizing a number of unique groups in the polymer backbone and their contributions to the resultant polymer features.;Several novel extended diether dianhydrides were synthesized. When these flexible dianhydrides were combined with rigid diamines, an alternating flexible/rigid polymer backbone resulted and hence the potential was created for liquid crystallinity. Other favorable components of liquid crystallinity, such as flexible spacers, rigid groups, and bulky groups, were incorporated into these novel dianhydrides and polymers therefrom. The potential liquid crystalline polyimides developed exhibited crystallinity and other desirable properties, but data were inconclusive regarding their liquid crystallinity. Extensive knowledge was gained in the synthesis of novel dianhydrides and their precursors. Additionally, structure-property relationships based on a variety of novel dianhydride moieties resulted.;High performance polymer film and coating materials are increasingly being used by the electronic circuit industry. Electrical behavior is critical for polymers used in these applications. Materials are needed with substantially lower dielectric constants. Fluorinated dianhydrides and polyimides therefrom were synthesized to achieve lower dielectric constants. Additionally, a series of polyimide copolymers were developed for use as semi-conductor stress relief layers, interlayer dielectrics, and encapsulants. Several combinations were achieved that optimized mechanical, physical, and chemical properties required for the applications.;Polyimides for use in harsh environments evaluated new and existing polyimides through physical, mechanical, and chemical means to determine possible substitutes for wire and cable insulation. These candidates have other utility in applications requiring hydrolytic stability. Also, squaric acid containing polyimides were developed and evaluated for potential space applications. These polyimides exhibited a combination of attractive properties, especially their resistance to the radiation component of geosynchronous orbit

The nature-printed British sea-weeds : a history, accompanied by figures and dissections, of the algae of the British Isles ; in four volumes

1. Rhodospermeae: fam. I. - IX. 2. Rhodospermeae: fam. X. - XIII. 3. Melanospermeae 4. Chlorospermea

Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19(INK4d)

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21(KIP1), for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of μ-calpain proteolysis of the p19(INK4d) protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: μ-calpain cleaves p19(INK4d) immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins

The calpains: modular designs and functional diversity

The eukaryotic calpains are a family of calcium-dependent papain-like proteases and their non-enzymatic relatives whose varied physiological functions are beginning to be fully explored

Over and over : consumer engagement and turning sports tourists into fans

Travel to consume sport is an increasingly popular and prevalent leisure pursuit (Fourie & Santana-Gallego, 2011; Fredline, 2005). From those who follow Formula 1 across the globe (Henderson et al., 2010), to Tennis’ most ardent fans (Fredline, 2005), consumers probe further afield in order to satisfy their desires for memorable and gratifying travel couched within the domain of their favourite sporting activities. Football consumption, although traditionally aligned along geographical or demographic boundaries (Jones, 2000; Porat, 2010, Conner, 2014), is no different with 800,000 overseas tourists travelling to the UK to experience matches every year (Magowan, 2015). The extent of this travel provides opportunities tangentially for service providers geographically proximate to major finals and international tournament destinations (Daniels, 2005; Prayag et al., 2013), but also at a granular level for football clubs who seek to attract, engage, and maintain access to this lucrative market of affluent consumers who spend over £680m annually (Magowan, 2015). Extant research is focused on the impact of this economic influx (Daniels, 2007; Smith, 2005, Allan et al., 2007), with little consideration given to the tourists themselves, nor the potential to engage with these one-off visitors in order to transform them into more regular sources of income. Therefore, engagement is crucial and, from an operational perspective, the football ‘industry’ has recognised this. The increasing prevalence of social media transfer announcements (Lang, 2017), innovations such as Manchester City’s newly developed glass-tunnel (Hyde 2017), and ‘city-takeover’ events aimed at bridging the gap between player-and-fan, demonstrate how football clubs are adopting unusual strategies in order to encourage consumers to believe that they have ‘behind-the-scenes’ access to the machinations of the clubs that they love. However, these attempts typically focus on local fans, neglecting those who travel to consumer sport on a regular basis. As such, the question remains, how can football clubs engage these tourists and encourage them to become ‘fans’ (through repeat visits and recommendations to friends) and thus benefit financially from their considerable spending power

MUTATION OF AN IQ-MOTIF: EVIDENCE FOR AN EXOSITE IN CALPAIN-2?

These studies aimed to test the hypothesis that calcium regulates calpain activity by a mechanism analogous to that used by Ca2+- calmodulin (CaM) to regulate its target enzymes. Site directed mutagenesis of a putative IQ motif, IQ413xxxR417 generated catalytic subunits modified at both, or each, of the key residues of the motif to yield ISxxxQ, ISxxxR, and IQxxxQ. Each of the mutant catalytic subunits was co-expressed with a truncated (21k) small subunit in E. coli. The heterodimeric enzymes were purified by standard methods suggesting each was properly folded. Electrophoresis under native conditions, in the absence of calcium, showed no differences between wt and enzyme mutated at R417Q. Each of these enzymes was also stable to incubation at 45 ºC. In contrast enzymes containing Q413S were resolved into multiple bands on native gels and were not stable at 45 ºC. Two calcium dependent activities were assessed: casein hydrolysis and autoproteolysis. Each of the mutants retained significant autolytic activity but lost much of their caseinolytic activity. We propose two interpretations of the results based in part on the calcium-free structures determined for calpain-2 (Hosfield et al , 1999 EMBO J. 18, 6880 and Strobl et al 2000, PNAS 97, 588). The ability of the enzymes to autoproteolyze suggests that the conformational change required for alignment of the catalytic residues is unimpaired in the mutants. Impaired casein hydrolysis may result from 1) inability to release product after cleavage or 2) failure to form a required substrate binding exosite. The latter concept is consistent with many previously known calpain attributes. Exosites provide a mechanism for achieving high substrate specificity amongst enzymes that share conserved active sites, such as those of the thrombin family and perhaps the calpain family. Confirmation and definition of such exosites will be important as they could provide targets for design of isoform selective inhibitors