Biomedical subjects

David B Williams

Publications and source records attributed to David B Williams.

17 recordsLinked to original sources

PubMed2006

Potent lectin-independent chaperone function of calnexin under conditions prevalent within the lumen of the endoplasmic reticulum.

Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.

Animals↗

PubMed2006

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules.

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.

Animals↗

PubMed2006

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum.

Calnexin and calreticulin are related proteins that comprise an ER chaperone system that ensures the proper folding and quality control of newly synthesized glycoproteins. The specificity for glycoproteins is conferred by a lectin site that recognizes an early oligosaccharide processing intermediate on the folding glycoprotein, Glc1Man9GlcNAc2. In addition, calnexin and calreticulin possess binding sites for ATP, Ca2+, non-native polypeptides and ERp57, an enzyme that catalyzes disulfide bond formation, reduction and isomerization. Recent studies have revealed the locations of some of these ligand-binding sites and have provided insights into how they contribute to overall chaperone function. In particular, the once controversial non-native-polypeptide-binding site has now been shown to function both in vitro and in cells. Furthermore, there is clear evidence that ERp57 participates in glycoprotein biogenesis either alone or in tandem with calnexin and calreticulin.

Animals↗

PubMed2006

In vitro assays of the functions of calnexin and calreticulin, lectin chaperones of the endoplasmic reticulum.

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum (ER) whose folding-promoting functions are directed predominantly toward aspargine-linked glycoproteins. This is a consequence of calnexin and calreticulin being lectins with specificity for the early oligosaccharide (OS)-processing intermediate, Glc1Man9GlcNAc2. In addition, they interact with non-native conformers of glycoprotein polypeptide chains to prevent aggregation and recruit the thiol oxidoreductase ERp57 to catalyze glycoprotein disulfide formation/isomerization. In vitro assays of these functions have contributed greatly to our understanding of how calnexin and calreticulin promote glycoprotein folding. This chapter describes the isolation of Glc1Man9GlcNAc2 OS, as well as the assay used to measure OS binding. Furthermore, details are provided of assays that detect ERp57 binding by calnexin and calreticulin, as well as the abilities of these chaperones to suppress the aggregation of non-native protein substrates.

Calnexin↗

PubMed2006

Assembly of MHC class I molecules within the endoplasmic reticulum.

MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.

Animals↗

PubMed2005

In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control.

Newly synthesized polypeptides entering the endoplasmic reticulum (ER) encounter a large array of molecular chaperones and folding factors that facilitate proper folding as well as assess folding status, retaining non-native proteins within the ER. Calnexin (CNX), an ER membrane protein, and its soluble homologue, calreticulin (CRT), are two important molecular chaperones that contribute to both processes. They are highly unusual chaperones in that they act as lectins, binding the Asn-linked oligosaccharides of newly synthesized glycoproteins, as well as recognizing the polypeptide segments of glycoproteins. Furthermore, they associate with ERp57, a thiol oxidoreductase, that is thought to enhance the oxidative folding of glycoproteins bound to CNX/CRT. These characteristics of CNX and CRT as well as their mode of action have been elucidated though the use of multiple in vitro and in vivo approaches. This chapter will focus on the description of a number of in vitro assays that have been used to characterize the lectin and ERp57-binding functions of CNX/CRT and also their abilities to act as molecular chaperones to suppress protein aggregation. In addition, we will describe insect and mammalian expression systems in which major histocompatibility complex class I molecules are used as model glycoprotein substrates for CNX and CRT. These systems have been valuable in assessing folding and quality control events in vivo that are influenced by CNX or CRT as well as in characterizing the spectrum of substrates that are recognized by these chaperones.

Animals↗

PubMed2005

A new TEM method of interfacial thin-film characterization.

Nano-scale thin films are of wide interest. The distribution of angles between the grain-boundary plane and the surfaces of such films can affect their mechanical behaviour. A transmission electron microscopy method is described to measure the distribution of such angles. This method is based on the well-known double-diffraction effect from two overlapping grains, but its implementation at multiple interfaces is made practically feasible by recent advances in computer and digital imaging techniques, as demonstrated in this paper.

Journal Article↗

PubMed2005

Delineation of the lectin site of the molecular chaperone calreticulin.

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.

Amino Acid Sequence↗

PubMed2004

Bap29/31 influences the intracellular traffic of MHC class I molecules.

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.

Animals↗

PubMed2003

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation.

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.

Animals↗

PubMed2003

Application of automated crystallography for transmission electron microscopy in the study of grain-boundary segregation.

Analytical Electron Microscopy (AEM) has brought significant progress in the study of grain-boundary segregation. Using X-ray energy-dispersive spectrometry (XEDS) in the AEM, elemental segregation information can be related to the crystallographic character of the same boundary via conventional Transmission Electron Microscope (TEM) diffraction techniques. While significant efforts have been made to improve XEDS analysis of sub-nanometer segregation layers, the methods for crystallographic characterization of grain boundaries have remained the same for several decades and labor-intensive processes. Recently, a method termed Automated Crystallography for TEM (ACT) was developed, which automates crystallographic characterization of grains under TEM observation. In the present work, we combine ACT and X-ray mapping via EDS in AEM for the study of Sb grain-boundary segregation in a rapidly solidified Cu-0.08 wt % Sb alloy. In contrast with previous reports, a large degree of anisotropy in Sb segregation level between different boundaries is found. ACT results suggest that one of the several grain boundaries observed with no detectable Sb segregation is very close to a Sigma 3 coincidence-site lattice structure. The reason for the observed anisotropy in the present alloy is discussed, based upon McLean's theory of segregation.

Journal Article↗

PubMed2003

Comparison of detectability limits for elemental mapping by EF-TEM and STEM-XEDS.

The analytical sensitivity in terms of the signal-to-noise ratio (SNR) was investigated for elemental mapping by a transmission electron microscope equipped with an energy filter (EF-TEM) and a scanning transmission electron microscope with an X-ray energy dispersive spectrometer (STEM-XEDS). To compare the detectability limits of the elemental maps by the two techniques, homogeneous Cu-0.98+/-0.34 wt% Mn and Cu-4.93+/-0.49 wt% Mn thin specimens were used. Elemental maps can be considered as either an image or a spectrum. Therefore, the detectability limits of the elemental maps were characterized by the spectral SNR. To evaluate the detectability limits of the elemental maps with statistical confidence limits such as 1 sigma, 2 sigma and 3 sigma, the SNR values were reviewed from the statistical point of view. In STEM-XEDS mapping, the spectral SNR values improve as the specimen thickness increases since the signal intensity increases. Conversely, the spectral SNR in EF-TEM mapping is maximized at a certain thickness and then reduces as the thickness increases. To compare the two mapping techniques with regard to the analytical sensitivity, a method to estimate the minimum mass fraction (MMF) from measured signal and background intensities was developed. In this experimental approach, the MMF value can be evaluated by selecting the appropriate SNR value corresponding to the statistical confidence limits. In comparing the estimated MMF values from the two mapping approaches, EF-TEM mapping can be more sensitive than STEM-XEDS mapping up to specimen thicknesses <20-30 nm in the 1 sigma confidence limit and < approximately 50 nm in the 3 sigma limits. However, as the specimen thickness increases, the XEDS maps provide better detectability limits in the Cu-Mn dilute alloy specimens.

Journal Article↗

PubMed2003

The structure of a metallic glass system using EXELFS and EXAFS as complementary probes.

The short-range atomic order around all three constituent atoms in a prototypical bulk metallic glass (BMG) system was probed in a complementary way, using extended X-ray absorption fine structure for neighborhood of the higher atomic number elements, and extended energy loss fine structure (EXELFS) for the lower atomic number ones. The Pd(x)Ni((80-x))P((20)) system is a prototype for a whole class of BMG formers which are 80% transition metal and 20% metalloid. We find that the structure of these BMGs could be explained in terms of those of glasses at the end of the BMG range, namely, Pd(60)Ni(20)P(20) and Pd(30)Ni(50)P(20). The binary phosphide crystals near [Formula: see text] and 80 are found to be simulate very well the local atomic structure of Pd(30)Ni(50)P(20) and Pd(60)Ni(20)P(20) glasses, respectively. The best glass former in this series, Pd(40)Ni(40)P(20), is best described by a weighted average of Pd(30)Ni(50)P(20) and Pd(60)Ni(20)P(20) structures.

Journal Article↗

PubMed2003

Histopathological and clinical evaluation of serrated adenomas of the colon and rectum.

We evaluated the diagnostic utility of the histological characteristics ascribed in the literature to serrated adenomas and developed a practical working model to allow their reliable identification. We also documented the frequency and location of serrated adenomas identified in an unselected series of individuals undergoing colonoscopic evaluation, as well as the clinical characteristics of those individuals. One hundred forty consecutive individuals (prospective polyp data set; 97 male, 43 female; age mean: 63.3 y; age range: 29-98 y) with 255 polyps were identified from 919 individuals undergoing colonoscopy. Further polyps previously removed from these individuals were added for the purpose of histological assessment (extended polyp data set, n = 380). All polyps were assessed by two independent examiners for eight selected architectural and cytological features of serrated adenomas. In the prospective polyp data set, 56 patients had 72 hyperplastic polyps, 7 had 9 serrated adenomas, 3 had 4 admixed polyps, and 98 had 170 conventional adenomas. There was no difference in the age, sex, or cancer association of the seven patients with serrated adenomas when compared with the case of other individuals with polyps. The prevalence of serrated adenomas was 9/919 (1%) in our population, with an average size of 5.8 mm. When assessing serrated adenomas histologically, the combination of nuclear dysplasia and serration of >/=20% of crypts provided the most accurate model for detection of these lesions (sensitivity 100%, specificity 97%). Other criteria provided supportive evidence but did not increase the diagnostic yield. The optimum model for the histological identification of the serrated adenoma includes the presence of a serrated architecture in >/=20% of crypts in association with surface epithelial dysplasia.

Adenoma↗

PubMed2002

Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin.

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.

Base Sequence↗

PubMed2002

Synthesis of a molecular mimic of the Glc1Man9 oligoside as potential inhibitor of calnexin binding to DeltaF508 CFTR protein.

Deletion of phenylalanine at position 508 of the CFTR protein is associated with a severe form of cystic fibrosis. Biosynthetic arrest of the misfolded DeltaF508 CFTR protein in the endoplasmic reticulum is due to prolonged interaction with protein chaperones. In order to overcome this retention and thereby restore the delivery of the protein to the plasma membrane, a molecular mimic of the glycoprotein oligoside moiety has been designed and synthesized. Ability of this mimic to inhibit the binding of the natural Glc1Man9GlcNAc oligoside to calnexin has been measured.

Calcium-Binding Proteins↗

PubMed2002

Mutant MHC class I molecules define interactions between components of the peptide-loading complex.

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗