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When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikrein-kinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates., Introduction The contact system is an enzymatic cascade in blood that becomes activated when blood contacts surface materials. For example, this occurs during blood sampling in a glass or plastic [...]

A natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] is examined to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. The results have shown that TRiC has an active role in rearranging the bound actin molecule.

A demonstration that mammalian cells secrete numerous transthyretin (TTR) disease-associated variants with wild-type efficiency in spite of compromised folding energetics, is presented. It is proposed that ER-assisted folding (ERAF) in competition with ER-associated degradation (ERAD) defines the unique secretory aptitude of each tissue.

Byline: James F. Knudsen (1,4), Uno Carlsson (2), Per Hammarstrom (2), Gerald H. Sokol (1,3), Louis R. Cantilena (1) Keywords: COX-2; celecoxib; carbonic anhydrase; bicarbonate; inflammation Abstract: Cyclooxygenase-2 (COX-2) is up-regulated in stromal and inflammatory cells. The inducible COX-2 isoform is expressed during inflammation, in some cancers, and in brain tissue after global and focal ischemia. Tissue acidosis is a dominant factor in inflammation, and contributes to pain and hyperalgesia. Recently, compelling epidemiological and clinical evidence has documented the COX-independent effects of some COX-2 inhibitors (i.e., celecoxib, valdecoxib, and rofecoxib) among these effects are carbonic anhydrase (CA) inhibition. Carbonic anhydrases are zinc metalloenzymes expressed in various cell types, including those of the kidney, where they act as general acid--base catalysts. The kidneys are also known to express the highest concentration of COX-2 messenger ribonucleic acid. Celecoxib, like the prototypic CA inhibitor acetazolamide, is structurally characterized by an unsubstituted sulfonamide moiety. In the present study, we report that celecoxib exhibits the characteristics of a potent CA inhibitor, showing inhibitory human carbonic anhydrase II (hCAII) activity in the nanomolar range. Valdecoxib was relatively less potent. Rofecoxib, which lacks the unsubstituted sulfonamide moiety characteristic of CA inhibitors, showed no significant hCAII inhibitory activity. The current study corroborates our earlier report of structure-activity relationships as predictors of such metabolic events as hyperchloremia, acidosis, and changes in calcium and phosphate disposition and clinical manifestations associated with CA inhibition reported with celecoxib. These data showing inhibition of hCAII by the unsubstituted sulfonamides celecoxib and valdecoxib, but not by rofecoxib, may have important implications for the elucidation of the mechanisms of action as well as the side effects associated with COX-2 inhibitors. Author Affiliation: (1) Division of Clinical Pharmacology and Medical Toxicology, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland (2) IFM-Department of Chemistry, Linkoping University, Linkoping, Sweden (3) New Hope Cancer Center, Hudson, Florida (4) Box 550, Smithsburg, MD, 21783 Article History: Registration Date: 01/01/2004

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases., Hundreds of human diseases, including the amyloidoses, are associated with protein misfolding (1-5). The 80 familial mutations that exacerbate [for example, [Val.sup.30] → [Met.sup.30] (V30M) and [Leu.sup.55] → [Pro.sup.55] (L55P) [...]

Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V1221), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V1221 TTR have nearly equivalent tetramer stabilities, V1221 cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance--presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.

Results indicate that stability of transthyretin tetramers is dependent on hydrophobic interactions and destabilization is brought about by electrostatic repulsions between two pairs of Lys15 and Lys15' amino acid residues implicating a role for the anion shielding in modulating stability and amyloid fibril formation.

Results show that amyloidogenicity of the soluble human plasma protein, transthyretin, is dependent on tertiary structural changes brought about by acid-mediated dissociation of the homotetramer into monomers. Data further indicate that tetramer to folded monomer transition can not form fibrils, suggesting a need for structural changes within the monomer.

Research has been conducted on the molten globule intermediate of bovine carbonic anhydrase II. The dependence of the stability or unfolding of this intermediate on metal ion cofactor has been investigated.

The use of an anionic oligothipphene derivative (p-FTAA) for detecting prefibrillar protein aggregates during in vitro fibrillation of three different amyloidogenic proteins is analyzed. The results have highlighted the use of p-FTAA as an additional tool for analyzing the process of protein aggregation.

A study related to the single-domain cyclophilin 18 (Cyp18), known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and also function as both a folding catalyst and a chaperone is presented. The chaperone effect of Cyp18 demonstrated for citrate synthase indicated a general chaperone effect of Cyp18 PPI.

Inhibitors based on p-aminobenzoylsulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators were employed to study a small-molecule stabilization of the exceptionally destabilized human carbonic anhydrase II (HCA II) mutant H107Y. The findings from the study could be used to design small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants which cause Marble brain disease (MBD).

The thermodynamics of destabilizing effects of the H107Y mutation, in human carbonic anhydrase II linked to marble brain disease (MBD) is demonstrated. The results suggest that the mutation is caused by long-range sterical destabilizing effects of the bulky tyrosine residue.

Studies about the specific and directed rearrangement of the [beta]-actin structure observed in the natural [beta]-acting-TRiC system, which is vital for guiding [beta]-actin to the native state is reported.

A study reports the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of tissue factor [(sub s)TF(sub R200W)] compared to that of the wild-type protein [(sub s)TF(sub wt)]. The study concludes that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate, both would contribute to decreased coagulation activity in TF(sub R200W) carriers.

A negatively charged conjugated polyelectrolyte is developed that displays different characteristic optical changes, detected visually or by adsorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. The formation of amyloid fibrils is reflected as an alteration of the geometry and the electronic structure of the bound polyelectrolyte chains and is detected by absorption and emission.

Changes of the optical properties of conjugated polyelectrolytes are utilized to monitor noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications. A regioregular, zwitterionic conjugated oligoelectrolyte is synthesized in order to create a probe with a defined set of optical properties and thus facilitates interpretation of biomolecule-oligoelectrolyte interactions.

Research has been conducted on D18G transthyretin. The authors describe the biophysical properties of this transthyretin, and report that it is an unstable monomeric protein.

The transthyretin (TTR) amyloid diseases, representative of numerous misfolding disorders, are of considerable interest because there are mutations that cause or suppress disease. The [Val.sup.30] → [Met.sup.30] (V30M) TTR mutation is the most prevalent cause of familial amyloid polyneuropathy in heterozygotes, whereas a [Thr.sup.119] → [Met.sup.119] (T119M) mutation on the second TTR allele protects V30M carriers from disease. Here, we show that the incorporation of one or more T119M TTR subunits into a predominantly V30M tetramer strongly stabilized the mixed tetramer against dissociation. Dissociation is required for amyloid formation, so these findings provide a molecular explanation for intragenic trans-suppression of amyloidosis. The data also suggest a potential therapeutic strategy, provide insight into tissue-specific deposition and amyloid composition, and support the validity of the amyloid hypothesis in human disease., The amyloidoses are a large group of protein misfolding diseases (1-3). The 80 transthyretin (TTR) amyloid diseases are representative of those where a full-length protein composes the fibrils. The TTR [...]

Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate HCA II cysteine mutants spin-labeled at selected positions to further study the interaction. Findings showed that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II had to be unfolded to approximately the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL.