Multilayer gyroid cubic membrane organization in green alga <Emphasis Type="Italic">Zygnema</Emphasis>

Biological cubic membranes (CM), which are fluid membranes draped onto the 3D periodic parallel surface geometries with cubic symmetry, have been observed within subcellular organelles, including mitochondria, endoplasmic reticulum, and thylakoids. CM transition tends to occur under various stress conditions; however, multilayer CM organizations often appear associated with light stress conditions. This report is about the characterization of a projected gyroid CM in a transmission electron microscopy study of the chloroplast membranes within green alga Zygnema (LB923) whose lamellar form of thylakoid membrane started to fold into multilayer gyroid CM in the culture at the end of log phase of cell growth. Using the techniques of computer simulation of transmission electron microscopy (TEM) and a direct template matching method, we show that these CM are based on the gyroid parallel surfaces. The single, double, and multilayer gyroid CM morphologies are observed in which space is continuously divided into two, three, and more subvolumes by either one, two, or several parallel membranes. The gyroid CM are continuous with varying amount of pseudo-grana with lamellar-like morphology. The relative amount and order of these two membrane morphologies seem to vary with the age of cell culture and are insensitive to ambient light condition. In addition, thylakoid gyroid CM continuously interpenetrates the pyrenoid body through stalk, bundle-like, morphologies. Inside the pyrenoid body, the membranes re-folded into gyroid CM. The appearance of these CM rearrangements due to the consequence of Zygnema cell response to various types of environmental stresses will be discussed. These stresses include nutrient limitation, temperature fluctuation, and ultraviolet (UV) exposure.     

Three-dimensional periodic cubic membrane structure in the mitochondria of amoebaeChaos carolinensis

Summary Through computer simulation of images produced by the transmission electron microscope (TEM), we have identified three-dimensional periodic cubic membrane structures in giant amoebae (Chaos carolinensis) mitochondria. The cubic membranes are based on the highly curved three-dimensional periodic cubic surfaces, sharing the same geometry of mathematically defined periodic minimal surfaces. The double-membrane structures identified here divide space into three separate and convoluted subspaces. Specimen preparation, specifically the tendency to cut oblique sections, of this membrane crystal has added to the complexity of the resulting TEM projections and until now prevented researchers from recognizing them. It is the added complexity of the oblique sections, though, that allows us to match the TEM projection to a computer simulation of the same with confidence. In this study, formation of cubic membrane structures in amoeba mitochondria was found to be dependent on diet. The cubic structures only occurred in the absence of food, and disappeared in the presence of food, suggesting a structural adaptation and possible advantages for amoeba's survival in nature. The verification of mathematically well-defined structures in unfed amoeba mitochondria is also important to the understanding of the mitochondrial bioenergetics in relation to the topology of the inner membrane, where major cellular energy production as well as free-radical generation are taking place. This understanding may carry great impact upon human health as far as aging and age-related degenerative diseases are concerned, especially as mitochondrial disorders have been implicated in these processes.Abbreviations G gyroid - D double diamond - P primitive - TEM transmission electron microscopy - PCS periodic cubic surface     

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.     

Morphological models of trabecular bone suitable for high-porosity regions and vertebrae

The complex microarchitecture of trabecular bone makes it difficult to perform computational analyses on the real structure. Researchers have often resorted to using morphological idealizations employing simplified geometries. One such idealized structure, based on the gyroid, was found to mimic trabecular bone well. However, structures generated using the basic gyroid equation manifested discontinuities at high porosities. Another disadvantage of the gyroid is that it cannot model vertebral trabecular bone, which generally resembles cubic cells. To address these two shortcomings, we describe: (i) a modified structure based on the skeletal gyroid, which remains connected even at extremely high porosities, (ii) a cubic grid-like structure, based on the primitive minimal surface, for studying vertebrae.     

Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

Conformational reorganization of the SARS coronavirus spike following receptor binding: implications for membrane fusion

The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.     

Glycosylation of pea cotyledon membranes

Pea cotyledons were injected with d-[(14)C]mannose or d-[(14)C]-glucosamine and incubated for 1 to 1.5 hours. Cotyledons were homogenized and subcellular fractions were isolated by differential centrifugation followed by linear sucrose density gradient centrifugation.Radioactivity that was precipitated by trichloroacetic acid was associated most extensively with rough endoplasmic reticulum, Golgi membranes, a membrane with a density of 1.14 grams per cubic centimeter (possibly plasma membrane) and an unidentified subcellular component with a density of 1.22 grams per cubic centimeter. Lower levels of incorporation were observed in protein bodies and mitochondria.Isolated membrane fractions were lipid-extracted to determine which components of the membrane contained the label. Rough endoplasmic reticulum contained the most extensively labeled lipids which had similar properties to the lipid intermediates thought to be involved in glycoprotein assembly. The lipid free residues of the various membrane fractions contained radioactivity that was released by protease treatment. Acid hydrolysis of the residues indicated that most of the radioactivity was associated with mannose or glucosamine. It appears that various subcellular components of the pea cotyledon possess glycoproteins that contain mannose and glucosamine.     

SARS冠状病毒PLpro蛋白酶的结构与功能

SARS冠状病毒基因组编码2种病毒蛋白酶,即木瓜样蛋白酶（PLpro）和3C样蛋白酶(3CLpro).其中,PLpro蛋白酶结构与功能研究是近年来冠状病毒分子生物学研究的热点之一. PLpro蛋白酶参与SARS冠状病毒1a(1ab)复制酶多聚蛋白N端部分的切割加工,是SARS冠状病毒复制酶复合体(RC)形成的重要调节蛋白分子;最新研究表明,SARS冠状病毒PLpro蛋白酶是一种病毒编码的去泛素化酶（DUB）,对细胞蛋白具有明显去泛素化作用;而且对泛素(Ub)和泛素样分子ISG15均具有活性. PLpro蛋白酶对宿主抗病毒天然免疫反应具有负调节作用,是SARS冠状病毒的一种重要干扰素拮抗分子.PLpro蛋白酶是一种多功能病毒蛋白酶.本文结合作者课题组研究工作,对SARS冠状病毒PLpro蛋白酶结构和功能研究最新进展进行综述.     

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.     

Cubic membrane structure in amoeba (Chaos carolinensis) mitochondria determined by electron microscopic tomography.

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae-matrix organization with mitochondrial activity.     

Auto-accumulation method using simulated annealing enables fully automatic particle pickup completely free from a matching template or learning data

Single-particle analysis is a 3-D structure determining method using electron microscopy (EM). In this method, a large number of projections is required to create 3-D reconstruction. In order to enable completely automatic pickup without a matching template or a training data set, we established a brand-new method in which the frames to pickup particles are randomly shifted and rotated over the electron micrograph and, using the total average image of the framed images as an index, each frame reaches a particle. In this process, shifts are selected to increase the contrast of the average. By iterated shifts and further selection of the shifts, the frames are induced to shift so as to surround particles. In this algorithm, hundreds of frames are initially distributed randomly over the electron micrograph in which multi-particle images are dispersed. Starting with these frames, one of them is selected and shifted randomly, and acceptance or non-acceptance of its new position is judged using the simulated annealing (SA) method in which the contrast score of the total average image is adopted as an index. After iteration of this process, the position of each frame converges so as to surround a particle and the framed images are picked up. This method is the first unsupervised fully automatic particle picking method which is applicable to EM of various kinds of proteins, especially to low-contrasted cryo-EM protein images.     

Computer-aided microtomography with true 3-D display in electron microscopy

A novel research system has been designed to permit three-dimensional (3-D) viewing of high resolution image data from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The system consists of front-end primary data acquisition devices, such as TEM and SEM machines, which are equipped with computer-controlled specimen tilt stages. The output from these machines is in analogue form, where a video camera attached to the TEM provides the sequential analogue image output while the SEM direct video output is utilized. A 10 MHz digitizer transforms the video image to a digital array of 512 X 512 pixel units of 8 bits deep-stored in a frame buffer. Digital images from multiple projections are reconstructed into 3-D image boxes in a dedicated computer. Attached to the computer is a powerful true 3-D display device which has hardware for graphic manipulations including tilt and rotate on any axis and for probing the image with a 3-D cursor. Data editing and automatic contouring functions are used to enhance areas of interest, and specialized software is available for measurement of numbers, distances, areas, and volumes. With proper archiving of reconstructed image sequences, a dynamic 3-D presentation is possible. The microtomography system is highly versatile and can process image data on-line or from remote sites from which data records would typically be transported on computer tape, video tape, or floppy disk.     

Novel cell culture device enabling three-dimensional cell growth and improved cell function

A better understanding of cell biology and cell-cell interactions requires three-dimensional (3-D) culture systems that more closely represent the natural structure and function of tissues in vivo. Here, we present a novel device that provides an environment for routine 3-D cell growth in vitro. We have developed a thin membrane of polystyrene scaffold with a well defined and uniform porous architecture and have adapted this material for cell culture applications. We have exemplified the application of this technology by growing HepG2 liver cells on 2- and 3-D substrates. The performance of HepG2 cells grown on scaffolds was significantly enhanced compared to functional activity of cells grown on 2-D plastic. The incorporation of thin membranes of porous polystyrene to create a novel device has been successfully demonstrated as a new 3-D cell growth technology for routine use in cell culture.     

Differential membrane binding of the human immunodeficiency virus type 1 matrix protein.

The human immunodeficiency virus type 1 matrix protein (p17MA) plays a central role at both the early and late stages of the virus life cycle. During viral assembly, the p17MA domain of Pr55gag promotes membrane association, which is essential for the formation of viral particles. When viral infection occurs, the mature p17MA dissociates from the plasma membrane and participates in the nuclear targeting process. Thus, p17MA contains a reversible membrane binding signal to govern its differential subcellular localization and biological functions. We previously identified a membrane binding signal within the amino-terminal 31 amino acids of the matrix domain of human immunodeficiency virus type 1 Gag, consisting of myristate and a highly basic region (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). Here we show that exposure of this membrane binding signal is regulated in different Gag protein contexts. Within full-length Pr55gag, the membrane targeting signal is exposed and can direct Pr55gag as well as heterologous proteins to the plasma membrane. However, in the context of p17MA alone, this signal is hidden and unable to confer plasma membrane binding. To investigate the molecular mechanism for regulation of membrane binding, a series of deletions within p17MA was generated by sequentially removing alpha-helical regions defined by the nuclear magnetic resonance structure. Removal of the last alpha helix (amino acids 97 to 109) of p17MA was associated with enhancement of binding to biological membranes in vitro and in vivo. Liposome binding experiments indicated that the C-terminal region of p17MA exerts a negative effect on the N-terminal MA membrane targeting domain by sequestering the myristate signal. We propose that mature p17MA adopts a conformation different from that of the p17MA domain within Pr55gag and present evidence to support this hypothesis. It is likely that such a conformational change results in an N-terminal myristyl switch which governs differential membrane binding.     

Cubic Membrane Structure in Amoeba (Chaos carolinensis) Mitochondria Determined by Electron Microscopic Tomography

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae–matrix organization with mitochondrial activity.     

Influence of hydrophobic and electrostatic residues on SARS‐coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS‐coronavirus (SARS‐CoV). SARS‐CoV entry is facilitated by the spike protein (S), which consists of an N‐terminal domain (S1) responsible for cellular attachment and a C‐terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS‐CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site‐directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH‐independent retroviral fusion proteins, SARS‐CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS‐CoV S2 analysis showed that specific hydrophobic positions at the C‐terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C‐terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C‐terminal hydrophobic residues led us to identify a 42‐residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.     

Identification of the MT3 molecule using two-dimensional gel electrophoresis and alloantisera

The MT3 antigen is defined serologically as a DR supertypic specificity and is strongly associated with DR4, DR7, and DRw9. To determine whether the MT3 molecule is distinct from the DR molecule, DR4 and MT3 antigens were immunoprecipitated from 125I-labeled plasma membrane glycoproteins of a DR4-homozygous, MT3-homozygous B lymphoid cell line, Wa, and compared by two-dimensional (2-D) gel electrophoresis. The precipitates with two different anti-DR4 alloantisera and with three different mouse antibodies against human Ia monomorphic determinants gave the same 2-D gel pattern consisting of one heavy chain with a molecular weight of 34 000 and a set of light chains with a molecular weight of 30 000, indicating that these polypeptides are the components of the DR4 molecule. On the other hand, all three anti-MT3 alloantisera used precipitated an identical set of anti-MT3 alloantisera specific light chains with a molecular weight of 30 000, and one heavy chain with a molecular weight of 34 000. The pI of the MT3 light chain was more acidic than that of the DR4 light chain. The amount of MT3 light chains was much smaller than that of DR4 light chains in unlabeled plasma membrane glycoproteins. Thus, we have demonstrated directly using 2-D gel electrophoresis and anti-MT3 alloantisera that the MT3 antigen is a new human Ia molecule distinct from DR4.     

Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus