Could Egg White Lysozyme be Solved by Single Particle Cryo-EM?

The combination of high-end cryogenic transmission electron microscopes (cryo-EM), direct electron detectors, and advanced image algorithms allows researchers to obtain the 3D structures of much smaller macromolecules than years ago. However, there are still major challenges for the single-particle cryo-EM method to achieve routine structure determinations for macromolecules much smaller than 100 kDa, which are the majority of all plant and animal proteins. These challenges include sample characteristics such as sample heterogeneity, beam damage, ice layer thickness, stability, and quality, as well as hardware limitations such as detector performance, beam, and phase plate quality. Here, single particle data sets were simulated for samples that were ideal in terms of homogeneity, distribution, and stability, but with realistic parameters for ice layer, dose, detector performance, and beam characteristics. Reference data were calculated for human apo-ferritin using identical parameters reported for an experimental data set downloaded from EMPIAR. Processing of the simulated data set resulted in a value of 1.86 Šfrom 20 214 particles, similar to a 2 Šdensity map obtained from 29 224 particles selected from real micrographs. Simulated data sets were then generated for a 14 kDa protein, hen egg white lysozyme (HEWL), with and without an ideal phase plate (PP). Whereas we could not obtain a high-resolution 3D reconstruction of HEWL for the data set without PP, the one with PP resulted in a 2.78 Šresolution density map from 225 751 particles. Our simulator and simulations could help in pushing the size limits of cryo-EM.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Evaluation of the impact of direct plating, broth enrichment, and specimen source on recovery and diversity of methicillin-resistant Staphylococcus aureus isolates among HIV-infected outpatients.

We compared recovery of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus (MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P ≤ 0.001) or MSA (P ≤ 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P ≤ 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P ≤ 0.001) and MSA (P ≤ 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P = 0.05).

Methicillin-resistant Staphylococcus aureus colonization in HIV-infected outpatients is common and detection is enhanced by groin culture.

SUMMARYAlthough high rates of clinical infection with methicillin-resistant Staphylococcus aureus (MRSA) have been reported in HIV-infected adults, data on MRSA colonization are limited. We enrolled HIV-infected adults receiving care at the Atlanta VA Medical Center. Swabs from each participant's nares and groin were cultured with broth enrichment for S. aureus. Of 600 HIV-infected adults, 79 (13%) were colonized with MRSA and 180 (30%) with methicillin-susceptible S. aureus. MRSA pulsed-field gel electrophoresis types USA300 (n=44, 54%) and USA500/Iberian (n=29, 35%) predominated. Inclusion of groin swabs increased MRSA detection by 24% and USA300 detection by 38%. In multivariate analysis, MRSA colonization compared to no MRSA colonization was associated with a history of MRSA clinical infection, rarely or never using condoms, and contact with prisons and jails. In summary, the prevalence of MRSA colonization was high in this study of HIV-infected adults and detection of USA300 was enhanced by groin culture.

Nevirapine-associated hepatotoxicity was not predicted by CD4 count ≥250 cells/µL among women in Zambia, Thailand and Kenya.

OBJECTIVE: The aim of the study was to determine risk factors for developing severe hepatotoxicity (grade 3 or 4 hepatotoxicity) and rash-associated hepatotoxicity (rash with ≥ grade 2 hepatotoxicity) among women initiating nevirapine-based antiretroviral therapy (ART). METHODS: The Non-Nucleoside Reverse Transcriptase Inhibitor Response Study was a prospective cohort study carried out in Zambia, Thailand and Kenya. Between May 2005 and January 2007, we enrolled antiretroviral-naïve HIV-infected women initiating nevirapine-based ART. At enrollment and at weeks 2, 4, 8, 16 and 24, participants had serum alanine transferase (ALT) and aspartate transaminase (AST) measured and were evaluated clinically for hepatitis and rash. RESULTS: Nevirapine-based ART was initiated in 820 women and baseline ALT or AST results were abnormal (≥ grade 1) in 113 (14%) women. After initiating nevirapine-based ART, severe hepatotoxicity occurred in 41 (5%) women and rash-associated hepatotoxicity occurred in 27 (3%) women. In a multivariate logistic regression model, severe hepatotoxicity and rash-associated hepatotoxicity were both associated with baseline abnormal (≥ grade 1) ALT or AST results, but not with a baseline CD4 cell count ≥250 cells/µL. Three participants (0.4%) died with symptoms suggestive of fatal hepatotoxicity; all three women had baseline CD4 count <100 cells/µL and were receiving anti-tuberculosis therapy. CONCLUSION: Among women taking nevirapine-based ART, severe hepatotoxicity and rash-associated hepatotoxicity were predicted by abnormal baseline ALT or AST results, but not by a CD4 count ≥250 cells/µL. In resource-limited settings where transaminase testing is available, testing should focus on early time-points and on women with abnormal baseline ALT or AST results.

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr.

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60-kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure. The frozen-hydrated sections were generated by sectioning at -150 degrees C using a diamond knife with a 25 degrees knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.

Characterization of coated vesicles that participate in endocytic recycling.

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.

The p41 isoform of invariant chain is a chaperone for cathepsin L.

The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41-CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack p41. This defect mainly concerns the mature two-chain forms of CatL, which depend on p41 to be expressed at wild-type levels. Indeed, pulse-chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.

CD1c molecules broadly survey the endocytic system.

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide.

A crucial feature of peptide antigen presentation by major histocompatibility complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens. Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol. In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules. Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome-lysosome fusion that may compromise the MHC class II pathway of antigen presentation. Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins. CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered. These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation. These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.

Dominance of immunoglobulin G2c in the antiphosphorylcholine response of rats infected with Trichinella spiralis.

The antibody response to the L1 stage of Trichinella spiralis has been described as biphasic. Worms resident in the intestine during the first week of infection stimulate an antibody response against a subset of larval proteins. L1 larvae in the muscle at the end stage of infection stimulate a second antibody response against tyvelose-bearing glycoproteins. Antityvelose antibodies protect rats against challenge infection with larvae. The aim of this study was to characterize the rat B-cell response against larval antigens during the intestinal phase of T. spiralis infection and to test the antiparasitic effects of such antibodies. Strain PVG rats were infected orally with 500 larvae. Antibodies specific for phosphorylcholine-bearing proteins of L1 larvae first appeared in serum 9 days postinfection. Absorption experiments showed that the majority of antilarval antibodies produced in rats 16 days after infection with T. spiralis were specific for phosphorylcholine-bearing proteins. A fraction of these antibodies bound to free phosphorylcholine. Immunoglobulin G2c (IgG2c) producing cells in the mesenteric lymph node dominated this early antibody response. IgG2c is associated with T-independent immune responses in the rat; however, a comparison of athymic rats with euthymic controls suggested that only a small fraction of the phosphorylcholine-related antibody response against T. spiralis was T independent. Phosphorylcholine is a common epitope in antigens of bacteria and nematode parasites and has been shown to be a target of protective immunity in certain bacteria. A monoclonal IgG2c antibody was prepared from infected rats and shown to be specific for phosphorylcholine. Monoclonal phosphorylcholine-specific IgG2c failed to protect rats against intestinal infection with T. spiralis. Therefore, our findings do not support a role for phosphorylcholine-bearing antigens in immune defense against T. spiralis; however, the potency of the immune response induced suggests an immunomodulatory role for the lymphocytes involved.

EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization.

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.

Separate pathways for antigen presentation by CD1 molecules.

The ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast.

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.