The environmental dependency of protein folding best explains prion and amyloid diseases.
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Alzheimer Disease↗
Biomedical subjects
J W Kelly
Publications and source records attributed to J W Kelly.
Recombinant human retinol-binding protein refolding, native disulfide formation, and characterization.
Human retinol-binding protein (RBP) is a monomeric 21-kDa protein that is currently the subject of numerous studies owing to its role in the cellular uptake and utilization of retinol. When the RBP gene is overexpressed in Escherichia coli, inclusion bodies of aggregated RBP are found in the cells. These inclusion bodies are solubilized in 5.0 M GdmCl containing 10 mM DTT. Refolding of RBP is carried out in the presence of vitamin A by diluting denatured and reduced RBP into a redox refolding buffer consisting of 3 mM cysteine/0.3 mM cystine at 4 degreesC. Ion exchange chromatography (HPLC) is utilized to purify refolded RBP to homogeneity as demonstrated by SDS-PAGE and electrospray MS. The native structure of refolded RBP was established by its ability to bind to vitamin A and the plasma protein transthyretin. The reconstitution of RBP outlined within affords a 50-60% overall yield, i.e., 73 mg of pure RBP/L of E. coli culture.
Chromatography, Ion Exchange↗
The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways.
The conformational change hypothesis postulates that tertiary structural changes under partially denaturing conditions convert one of 17 normally soluble and functional human proteins into an alternative conformation that subsequently undergoes self-assembly into an amyloid fibril, the putative causative agent in amyloid disease. This hypothesis is consistent with Anfinsen's view that the tertiary structure of a protein is determined both by its sequence and the aqueous environment; the latter does not always favor the normally folded state. Unlike sickle cell hemoglobin assembly, where owing to a surface mutation, hemoglobin polymerizes in its normally folded conformation, amyloid proteins self-assemble as a result of the formation of an alternative tertiary structure-a conformational intermediate formed under partially denaturing conditions. The pathway by which an amyloidogenic protein assembles into amyloid fibrils appears to involve quaternary structural intermediates that assemble into increasingly complex quaternary structures, including amyloid protofilaments, which ultimately assemble into amyloid fibrils. Several recent studies have discussed the multi-step assembly pathway(s) characterizing amyloid fibril formation.
Amyloid↗
Discovering transthyretin amyloid fibril inhibitors by limited screening.
Insoluble protein fibrils, resulting from the self-assembly of a conformational intermediate are implicated to be the causative agent in several human amyloid diseases including familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). These diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of a lysosome or endosome. Here we identify several structural classes of small molecules that are capable of inhibiting the TTR conformational changes facilitating amyloid fibril formation. A small molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a promising approach to treat amyloid diseases and to rigorously test the amyloid hypothesis, the apparent causative role of amyloid fibrils in amyloid disease.
Amyloid↗
Once weekly azithromycin therapy for prevention of Mycobacterium avium complex infection in patients with AIDS: a randomized, double-blind, placebo-controlled multicenter trial.
We conducted a randomized, double-blind, placebo-controlled multicenter trial of azithromycin (1,200 mg once weekly) for the prevention of Mycobacterium avium complex (MAC) infection in patients with AIDS and a CD4 cell count of < 100/mm3. In an intent-to-treat analysis through the end of therapy plus 30 days, nine (10.6%) of 85 azithromycin recipients and 22 (24.7%) of 89 placebo recipients developed MAC infection (hazard ratio, 0.34; P = .004). There was no difference in the ranges of minimal inhibitory concentrations of either clarithromycin or azithromycin for the five breakthrough (first) MAC isolates from the azithromycin group and the 18 breakthrough MAC isolates from the placebo group. Of the 76 patients who died during the study, four (10.5%) of 38 azithromycin recipients and 12 (31.6%) of 38 placebo recipients had a MAC infection followed by death (P = .025). For deaths due to all causes, there was no difference in time to death or number of deaths between the two groups. Episodes of non-MAC bacterial infection per 100 patient years occurred in 43 azithromycin recipients and 88 placebo recipients (relative risk, 0.49; 95% confidence interval, 0.33-0.73). The most common toxic effect noted during the study was gastrointestinal, reported by 78.9% of azithromycin recipients and 27.5% of placebo recipients. Azithromycin given once weekly is safe and effective in preventing disseminated MAC infection, death due to MAC infection, and respiratory tract infections in patients with AIDS and CD4 cell counts of < 100/mm3.
AIDS-Related Opportunistic Infections↗
Characteristics of familial and non-familial melanoma in Australia.
Between 6 and 14% of malignant melanomas have been reported to occur in a familial pattern. In this study 785 melanoma patients from the Victorian Melanoma Service and the private practice of a dermatologist were assessed for the total number of melanocytic naevi, the number of dysplastic naevi and other clinical characteristics categorized according to whether there was a family history of melanoma. It was found that the presence of 100 or more naevi, six or more dysplastic naevi and blue eyes in a patient with melanoma were significantly associated with a family history of melanoma. Patients with two or more family members with melanoma were significantly more likely to develop melanoma at a younger age and to develop multiple melanomas. This study concludes that large numbers of melanocytic naevi and dysplastic naevi in melanoma patients are useful characteristics in practice to identify patients at risk of familial occurrence of melanoma. Family members of these patients should receive a medical assessment for their pigmented lesions.
Adult↗
Familial Kaposi's sarcoma and Paget's disease of bone.
Familial Kaposi's sarcoma and familial Paget's disease of bone have not previously been reported to occur in the one patient or the one family. We report on an 82-year-old female of Lebanese descent who was recently diagnosed with Kaposi's sarcoma and Paget's disease. Of the patient's eight siblings, seven had Paget's disease and two of these also had Kaposi's sarcoma. Histocompatibility leucocyte antigen (HLA) class I and II typing of the patient showed: A2, A3; B35, Bx; Bw6; Cw4; DR beta 1*1101 (an HLA-DR5 subtype), DR beta 3 and DQ beta 1*0301. Previous reports have described possible associations of familial Kaposi's sarcoma with HLA-DR5 and Paget's disease with DR2. DR beta 1*1104, DP beta 1*04 and DQw1. Genetic factors and possible viral aetiologies for each condition are reviewed.
Aged↗
Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis: equilibria with high kinetic barriers.
Fluorescence and circular dichroism spectroscopy as well as analytical ultracentrifugation and glutaraldehyde cross-linking were utilized to evaluate the tertiary and quaternary structural changes occurring on the denaturation and reconstitution pathways of transthyretin (TTR) as a function of guanidine hydrochloride (GdnHCl) concentration. These results demonstrate that the GdnHCl-mediated denaturation and reconstitution of TTR is reversible. However, the lowest GdnHCl concentration that dissociates and unfolds transthyretin does not allow the unfolded monomer to refold to tetramer at a rate that is measurable. As a result, there is a striking hysteresis observed upon comparison of the GdnHCl-mediated denaturation and reconstitution transitions. The TTR tetramer does not dissociate into unfolded monomer until the denaturant concentration exceeds 4 M GdnHCl, whereas unfolded monomeric TTR (denatured in 7 M GdnHCl) does not refold and assemble into a native tetrameric structure until the GdnHCl concentration is reduced to less than 2 M. These results imply that a significant kinetic barrier intervenes between the folded tetramer and unfolded monomer in both the denaturation and reconstitution directions at pH 7. A kinetics study of the denaturation of TTR as a function of GdnHCl concentration yields a first-order rate constant for unfolding of (9.0 +/- 7.5) x 10(-11) s-1, estimated by extrapolation of the rate constants for the tetramer to unfolded monomer transition as a function of GdnHCl to 0 M GdnHCl. This rate is very slow; as a result, wild-type TTR is predicted to be kinetically stable as a tetrameric quaternary structure once formed. These results imply that the rate of TTR dissociation and partial unfolding to the monomeric amyloidogenic intermediate under denaturing conditions may play a role in transthyretin-based amyloid diseases.
Circular Dichroism↗
A high incidence of melanoma found in patients with multiple dysplastic naevi by photographic surveillance.
OBJECTIVES: (1) To assess the incidence of melanoma in a cohort of patients with dysplastic melanocytic naevi (DMN) and the relationships between incident melanomas and preexisting naevi and between melanoma risk and numbers of DMN. (2) To examine the role of the patient versus the physician in detecting melanoma and the relative value of surveillance versus prophylactic excision. DESIGN: Prospective cohort study. PATIENTS AND SETTING: Two hundred and seventy-eight adults, each with five or more DMN, were followed up for a mean period of 42 months in a private dermatology practice. DMN were clinically diagnosed. RESULTS: Twenty new melanomas were detected in 16 patients, corresponding to an age-adjusted incidence of 1835/100000 person-years, 46 times the incidence in the general population. Eleven were detected because of changes evident in comparison with baseline photographs and nine were detected by patients or their partners. Thirteen of the 20 melanomas arose as new lesions and only three from DMN. Melanoma risk rose with increasing numbers of DMN. CONCLUSIONS: Increasing numbers of DMN are associated with increasing melanoma risk. Surveillance (baseline photography and follow-up) enabled early diagnosis of melanoma and was very much more cost-effective in preventing life-threatening melanoma than prophylactic excision of DMN.
Adolescent↗
Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands.
We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domain-binding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using site-directed mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.
Adaptor Proteins, Signal Transducing↗
Amyloid fibril formation and protein misassembly: a structural quest for insights into amyloid and prion diseases.
The assembly and misassembly of normally soluble proteins into fibrilar structures is thought to be a causative agent in a variety of human amyloid and prion diseases. Structural and mechanistic studies of this process are beginning to elucidate the conformational changes required for the conversion of a normally soluble and functional protein into a defined quaternary structure.
Amyloid↗
Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone.
Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.
Arginine↗
Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid.
Human transthyretin (TTR) can be transformed into amyloid fibrils by partial acid denaturation to yield a monomeric amyloidogenic intermediate that self-associates into amyloid through quaternary structural intermediates, which are identified by sedimentation velocity methods. The monomeric amyloidogenic intermediate has substantial beta-sheet structure with a nonnative but intact tertiary structure as discerned from spectroscopic methods. Proteolysis sensitivity studies suggest that the C-strand-loop-D-strand portion of TTR becomes disordered and moves away from the core of the beta-sandwich fold upon formation of the monomeric amyloidogenic intermediate over the pH range 5.1-3.9. The single site mutations that are associated with early onset amyloid disease [familial amyloid polyneuropathy (FAP)] function by destabilizing tetrameric TTR. Under mild denaturing conditions, the FAP variants populate the monomeric amyloidogenic intermediate conformation, which assembles into amyloid, whereas wild-type TTR remains tetrameric and nonamyloidogenic. The FAP mutations do not significantly alter the native folded structure; instead, they appear to act by making the thermodynamics and perhaps the kinetics more favorable for formation of the amyloidogenic intermediate. Suppressor mutations have also been characterized that strongly stabilize tetrameric TTR and disfavor the formation of the monomeric amyloidogenic intermediate, thus inhibiting amyloid formation. The mechanistic details characterizing transthyretin amyloid fibril formation available from the biophysical studies outlined within have been utilized to develop a new therapeutic strategy for intervention in human amyloid disease. This approach features small molecules that bind with high affinity to the normal fold of transthyretin, inhibiting the quaternary and tertiary structural changes associated with the formation of the monomeric amyloidogenic intermediate that self-assembles into amyloid. Ligand binding to TTR stabilizes the native tetrameric fold, which is nonamyloidogenic.
Amyloid↗
Spitz naevi misdiagnosed histologically as melanoma: prevalence and clinical profile.
A Spitz naevus is a benign melanocytic tumour that may histologically resemble a malignant melanoma. Data was retrospectively gathered from patients who attended the Victorian Melanoma Service to determine the prevalence of Spitz naevi pathologically misdiagnosed as melanoma. Assessment of the clinical characteristics of these patients was also performed and compared to those with correctly diagnosed melanoma. It was found that 6.5% of all melanomas referred were in fact Spitz naevi and that Spitz naevi represented the majority of pathologically misdiagnosed melanomas. The Spitz naevi were more likely to be on the lower extremities and were no average, considerably smaller than the melanomas. Patients with Spitz naevi were more likely to be younger, female, have fewer dysplastic naevi and have brown eyes. One hundred per cent of the Spitz naevi were brought to the attention of the initial doctor by the patient compared to 72% of the melanomas. This study concludes that Spitz naevi that are pathologically misdiagnosed as melanomas retain the clinical characteristics of other Spitz naevi and that greater clinicopathological communication may reduce the frequency of diagnostic error.
Chi-Square Distribution↗
Differences in age and body site distribution of the histological subtypes of basal cell carcinoma. A possible indicator of differing causes.
OBJECTIVE: To assess if there are any significant differences in the sex, anatomical site, and age distribution of patients with different histological subtypes of basal cell carcinoma (BCC). DESIGN: Histopathology reports were analyzed with respect to the subtype of BCC, site of a tumor, and age and sex of a patient. SETTING: Histopathology reports were reviewed from 1 private laboratory that derived its cases from general practitioners, surgeons, and dermatologists. PATIENTS: Patients with BCC (N = 3885) for whom case data were received by the pathology laboratory from January 2, 1991, to June 12, 1991, were included in the study. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Superficial BCCs differed from other subtypes of BCC by occurring more commonly on the trunk and in younger patients. RESULTS: The mean age (56.8 years) of the patients with superficial BCCs was significantly lower than that of the patients with other subtypes of BCC who were examined (P < .001); the mean ages of the patients with these other subtypes were as follows: nodulosuperficial BCC, 62.9 years; nodular BCC, 63.9 years; nodulomorpheic BCC, 66.1 years; and morpheic BCC, 66.0 years. The majority of superficial BCCs occurred on the trunk and limbs (73.3%), while the majority of all other subtypes occurred on the head and neck. CONCLUSIONS: Superficial BCC differs from the other subtypes of BCC in terms of patient age and tumor site, and these findings may reflect differences in the etiology.
Adolescent↗
Inhibiting transthyretin amyloid fibril formation via protein stabilization.
Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 microM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis--i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.
Amyloidosis↗
The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid.
Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.
Amyloid↗
Malignant melanomas--how many have you missed?
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Adult↗