Biomedical subjects

Alasdair C Steven

Publications and source records attributed to Alasdair C Steven.

At least 55 records · Page 3Linked to original sources

PubMed2003

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber.

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.

Blotting, Western↗

PubMed2003

A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core.

Like other bacteriophages, T7 has a singular vertex that is the site of a symmetry mismatch involving the portal/connector protein, a 12-fold ring at the vertex site which is also a 5-fold axis for the icosahedral capsid. In the mature virion, a 6-fold-symmetric tail extends outwards from the connector. T7 also has a cylindrical "core" that assembles on the inner surface of the connector during procapsid formation, is retained in the mature virion, and is required for infectivity. We have investigated the core structure by cryo-electron microscopy and image analysis of procapsids and find that it observes 8-fold symmetry. Stoichiometry data indicate that its major constituent is an octamer of gp15.

Bacteriophage T7↗

PubMed2003

Molecular mechanisms in bacteriophage T7 procapsid assembly, maturation, and DNA containment.

Bacteriophage T7 is a double-stranded DNA bacteriophage that has attracted particular interest in studies of gene expression and regulation and of morphogenesis, as well as in biotechnological applications of expression vectors and phage display. We report here studies of T7 capsid assembly by cryoelectron microscopy and image analysis. T7 follows the canonical pathway of first forming a procapsid that converts into the mature capsid, but with some novel variations. The procapsid is a round particle with an icosahedral triangulation number of 7 levo, composed of regular pentamers and elongated hexamers. A singular vertex in the procapsid is occupied by the connector/portal protein, which forms 12-fold and 13-fold rings when overexpressed, of which the 12-mer appears to be the assembly-competent form. This vertex is the site of two symmetry mismatches: between the connector and the surrounding five gp 10 hexamers; and between the connector and the 8-fold cylindrical core mounted on its inner surface. The scaffolding protein, gp9, which is required for assembly, forms nubbin-like protrusions underlying the hexamers but not the pentamers, with no contacts between neighboring gp9 monomers. We propose that gp9 facilitates assembly by binding to gp10 hexamers, locking them into a morphogenically correct conformation. gp9 is expelled as the procapsid matures into the larger, thinner walled, polyhedral capsid. Several lines of evidence implicate the connector vertex as the site at which the maturation transformation is initiated: in vivo, maturation appears to be triggered by DNA packaging whereby the signal may involve interaction of the connector with DNA. In the mature T7 head, the DNA is organized as a tightly wound coaxial spool, with the DNA coiled around the core in at least four and perhaps as many as six concentric shells.

Bacteriophage T7↗

PubMed2003

Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding.

Electron cryotomography has unique potential for three-dimensional visualization of macromolecular complexes at work in their natural environment. This approach is based on reconstructing three-dimensional volumes from tilt series of electron micrographs of cells preserved in their native states by vitrification. Resolutions of 5-8 nm have already been achieved and the prospects for further improvement are good. Since many intracellular activities are conducted by complexes in the megadalton range with dimensions of 20-50 nm, current resolutions should suffice to identify many of them in tomograms. However, residual noise and the dense packing of cellular constituents hamper interpretation. Recently, tomographic data have been collected on vitrified eukaryotic cells (Medalia et al., Science (2002) in press). Their cytoplasm was found to be markedly less crowded than in the prokaryotes previously studied, in accord with differences in crowding between prokaryotic and eukaryotic cells documented by other (indirect) biophysical methods. The implications of this observation are twofold. First, complexes should be more easily identifiable in tomograms of eukaryotic cytoplasm. This applies both to recognizing known complexes and characterizing novel complexes. An example of the latter-a 5-fold symmetric particle is-given. Second, electron cryotomography offers an incisive probe to examine crowding in different cellular compartments.

Animals↗

PubMed2003

The next ice age: cryo-electron tomography of intact cells.

Recent advances in electron tomography are beginning to reveal the internal structure of eukaryotic cells in their native states in three dimensions at molecular resolution. These observations represent the culmination of years of effort to develop protocols for automated data collection, image reconstruction and cryogenic preservation. Cryo-tomograms of Dictyostelium cells depict distinct populations of ribosomes, proteasomes and networks of actin filaments interconnected by branching or bundling, apparently controlled by strategically placed actin-associated proteins.

Animals↗

PubMed2003

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy.

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.

Animals↗

PubMed2003

Human immunodeficiency virus type 1 N-terminal capsid mutants containing cores with abnormally high levels of capsid protein and virtually no reverse transcriptase.

We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

Capsid Proteins↗

PubMed2002

The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer.

Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.

Animals↗

PubMed2002

Alternating translocation of protein substrates from both ends of ClpXP protease.

In ClpXP protease complexes, hexameric rings of the ATP-dependent ClpX chaperone stack on one or both faces of the double-heptameric rings of ClpP. We used electron microscopy to record the initial binding of protein substrates to ClpXP and their accumulation inside proteolytically inactive ClpP. Proteins with N- or C-terminal recognition motifs bound to complexes at the distal surface of ClpX and, upon addition of ATP, were translocated to ClpP. With a partially translocated substrate, the non-translocated portion remained on the surface of ClpX, aligned with the central axis of the complex, confirming that translocation proceeds through the axial channel of ClpXP. Starting with substrate bound on both ends, most complexes translocated substrate from only one end, and rarely (<5%) from both ends. We propose that translocation from one side is favored for two reasons: initiation of translocation is infrequent, making the probability of simultaneous initiation low; and, further, the presence of protein within the cis side translocation channel or within ClpP generates an inhibitory signal blocking translocation from the trans side.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2002

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization.

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.

Adenosine Triphosphatases↗

PubMed2002

Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein.

The [URE3] infectious protein (prion) of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal "prion" domain is responsible for amyloid filament formation (prion conversion). On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p. We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein. All formed amyloid filaments in vitro, whose diameters increased with the mass of the appended enzyme. The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct. CD data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all beta-sheet. In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures. These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in [URE3] cells by a steric blocking mechanism.

Amyloid↗

PubMed2002

Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP.

Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.

Adenosine Triphosphatases↗

PubMed2002

The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface.

Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell-forming 'core' domain and the nucleic acid-binding 'protamine' domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain-containing capsids, although its size-determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo-EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence-similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.

Amino Acid Substitution↗

PubMed2002

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs.

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.

Amino Acid Sequence↗

PubMed2002

Quasi-normal cornified cell envelopes in loricrin knockout mice imply the existence of a loricrin backup system.

The cornified cell envelope, a lipoprotein layer that assembles at the surface of terminally differentiated keratinocytes, is a resilient structure on account of covalent crosslinking of its constituent proteins, principally loricrin, which accounts for up to 60%-80% of total protein. Despite the importance of the cell envelope as a protective barrier, knocking out the loricrin gene in mice results in only mild syndromes. We have investigated the epidermis and forestomach epithelium of these mice by electron microscopy. In both tissues, corneocytes have normal-looking cell envelopes, despite the absence of loricrin, which was confirmed by immunolabeling, and the absence of the distinctive loricrin-containing keratohyalin granules (L-granules). Isolated cell envelopes were normal in thickness (approximately 15 nm) and mass per unit area (approximately 7.3 kDa per nm2); however, metal shadowing revealed an altered substructure on their cytoplasmic surface. Their amino acid compositions indicate altered protein compositions. Analysis of these data implies that the epidermal cell envelopes have elevated levels of the small proline-rich proteins, and cell envelopes of both kinds contain other protein(s) that, like loricrin, are rich in glycine and serine. These observations imply that, in the absence of loricrin, the mechanisms that govern cell envelope assembly function normally but employ different building-blocks.

Amino Acids↗

PubMed2002

Handedness of the herpes simplex virus capsid and procapsid.

The capsid of herpes simplex virus has an icosahedral surface lattice with a nonskew triangulation number, T=16. Nevertheless, the proteins arrayed on this lattice necessarily have an intrinsic handedness. We have determined the handedness of both the herpes simplex virus type 1 capsid and its precursor procapsid by a cryoelectron microscopic tilting method.

Capsid↗

PubMed2002

mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53.

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

Animals↗

PubMedDate not supplied

Cryo-electron microscopy of trichocyte (hard alpha-keratin) intermediate filaments reveals a low-density core.

Trichocyte intermediate filaments (IF) are the principal components of epidermal appendages such as hair and nail. Based on studies by a variety of techniques, it has been inferred that trichocyte IF are structurally similar to other kinds of IF. However, some basic structural attributes have yet to be established: in particular, it has remained unclear whether IF are hollow. We have examined trichocyte IF isolated from rat vibrissae and human hair follicles by electron microscopy. Scanning transmission electron microscopy of freeze-dried specimens yielded mass-per-unit-length values of approximately 32 kDa/nm, with the human preparations also containing filaments at half this density, corresponding to two rather than four protofibrils. Radial density profiles calculated from cryo-electron micrographs of vitrified specimens preserved in a near-native state revealed a low-density region of approximately 3 nm diameter around the filament axis. A minor species of filament with the same internal structure was surface-decorated with material arranged with a helical pitch length of 9.3 nm. These filaments appear to represent IF coated with associated proteins-perhaps, "high-sulfur" proteins-readied for incorporation into the filament-matrix biocomposite of the mature hair.

Animals↗