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1.
After their co-translational insertion into the ER lumen or the ER
membrane, most proteins are transported via the Golgi apparatus downstream
on the secretory pathway while a few protein species are retained in the
ER. Polypeptide retention in the ER is either signal-independent or depends
on specific retention signals encoded by the primary sequence of the
polypeptide. A first category, i.e. the newly synthesized polypeptides that
are unable to reach their final conformation, are retained in the ER where
this quality control generally results in their degradation. A second
category, namely the ER-resident proteins escape the bulk flow of secretion
due to the presence of a specific N- or C-terminal signal that interacts
with integral membrane or soluble receptors. ER retention of soluble
proteins mediated by either KDEL, HDEL or related sequences and membrane
receptors has been relatively well characterized in plants. Recent efforts
has been relatively well characterized in plants. Recent efforts have aimed
at a characterization of the retention signal(s) of type I membrane
proteins in the plant ER. 相似文献
2.
As the number of complete genomes that have been sequenced keeps growing, unknown areas of the protein space are revealed and new horizons open up. Most of this information will be fully appreciated only when the structural information about the encoded proteins becomes available. The goal of structural genomics is to direct large-scale efforts of protein structure determination, so as to increase the impact of these efforts. This review focuses on current approaches in structural genomics aimed at selecting representative proteins as targets for structure determination. We will discuss the concept of representative structures/folds, the current methodologies for identifying those proteins, and computational techniques for identifying proteins which are expected to adopt new structural folds. 相似文献
3.
Lundstrom K 《Molecular biotechnology》2006,34(2):205-212
Structural genomics can be defined as structural biology on a large number of target proteins in parallel. This approach plays
an important role in modern structure-based drug design. Although a number of structural genomics initiatives have been initiated,
relatively few are associated with integral membrane proteins. This indicates the difficulties in expression, purification,
and crystallization of membrane proteins, which has also been confirmed by the existence of some 100 high-resolution structures
of membrane proteins among the more than 30,000 entries in public databases. Paradoxically, membrane proteins represent 60–70%
of current drug targets and structural knowledge could both improve and speed up the drug discovery process. In order to improve
the sucess rate for structure resolution of membrane proteins structural genomics networks have been established. 相似文献
4.
Recent years have seen the establishment of structural genomics centers that explicitly target integral membrane proteins. Here, we review the advances in targeting these extremely high-hanging fruits of structural biology in high-throughput mode. We observe that the experimental determination of high-resolution structures of integral membrane proteins is increasingly successful both in terms of getting structures and of covering important protein families, for example, from Pfam. Structural genomics has begun to contribute significantly toward this progress. An important component of this contribution is the set up of robotic pipelines that generate a wealth of experimental data for membrane proteins. We argue that prediction methods for the identification of membrane regions and for the comparison of membrane proteins largely suffice to meet the challenges of target selection for structural genomics of membrane proteins. In contrast, we need better methods to prioritize the most promising members in a family of closely related proteins and to annotate protein function from sequence and structure in absence of homology. 相似文献
5.
The preparation of large quantities of purified membrane proteins for structural studies presents significant difficulties. Central among these are the frequent toxicity associated with over-expressing membrane targets and the difficulty associated with identifying the appropriate detergents for their solubilization and purification. To begin addressing these challenges, and lay the groundwork for membrane structural genomics efforts, we have developed a robust strategy for the expression and purification of large numbers of prokaryotic membrane proteins. Our approach rapidly identifies highly expressed targets and greatly simplifies their solubilization and purification. In this review, specific, hands-on protocols are provided for the expression and purification of CorA magnesium transporters. These methods form the basis for the expression and purification of many other membrane proteins, as discussed. 相似文献
6.
7.
Kefala G Kwiatkowski W Esquivies L Maslennikov I Choe S 《Journal of structural and functional genomics》2007,8(4):167-172
Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their
study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have
addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration
levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo
proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental
for high-level expression of other integral membrane proteins. 相似文献
8.
Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world. 相似文献
9.
Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review. 相似文献
10.
Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca(2+) pump (SERCA) revealing different conformations, and recently, crystal structures of the H(+)-ATPase and the Na(+)/K(+)-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca(2+) signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na(+)/K(+)-ATPase, and other P-type ATPases. 相似文献
11.
12.
Kenneth Lundstrom 《生物化学与生物物理学报:生物膜》2003,1610(1):90-96
Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs. 相似文献
13.
Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins 总被引:1,自引:0,他引:1
Lundstrom K 《Biochimica et biophysica acta》2003,1610(1):90-96
Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs. 相似文献
14.
This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the
University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are
mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and
proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with
human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other
eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly
suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of
low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and
their dynamic properties during protein recognition. Several examples are given in this paper. 相似文献
15.
Libusha Kelly Ursula Pieper Narayanan Eswar Franklin A. Hays Min Li Zygy Roe-Zurz Deanna L. Kroetz Kathleen M. Giacomini Robert M. Stroud Andrej Sali 《Journal of structural and functional genomics》2009,10(4):269-280
Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth
and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein
families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human
G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane
proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to
define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins
belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully
cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1)
to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily,
(2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane
protein families. 相似文献
16.
Fleming KG 《Current opinion in biotechnology》2000,11(1):67-71
Genome sequencing efforts have revealed that perhaps as many as 20-40% of open reading frames in complex organisms may encode proteins containing at least one helical transmembrane segment. Contrasting with this approaching tidal wave of helical membrane proteins is the fact that our understanding of the sequence-structure-function relationships for membrane proteins lags far behind that of soluble proteins. This looming reality emphasizes the tremendous biochemical and structural work that remains to be done on helical membrane proteins in order to elucidate the structural and energetic principles that specify and stabilize their folds, which define their functions. These facts are not lost on the pharmaceutical industry, where successful therapeutics and major discovery efforts are targeting membrane proteins. 相似文献
17.
The nuclear envelope is a complex structure consisting of nuclear membranes, nuclear pore complexes and lamina. Several integral membrane proteins specific to the nuclear pore membrane and the inner nuclear membrane are known. Pore membrane proteins are probably important for organization and assembly of the nuclear pore complex, while proteins of the inner nuclear membrane are likely to play major roles in the structure and dynamics of the nuclear lamina and chromatin. Biochemical studies are now identifying potential binding partners for some of these integral membrane proteins, and analysis of nuclear envelope assembly at the end of mitosis is providing important insights into their functions. 相似文献
18.
Summary The purpose of this review is to summarize recent progress in understanding interactions of spectrin with membranes from brain and other tissues. Spectrin has at least two choices in linkages with the membrane, one through ankyrin, which in turn is associated with integral membrane proteins, and another linkage directly with integral membrane sites identified recently in brain membranes. Some of the integral membrane protein sites in brain bind preferentially with one spectrin isoform, while some can interact with both erythroid and the general isoform of spectrin. Ankyrin also has different isoforms, and these exhibit specificity in binding to spectrin isoforms and associate with distinct integral membrane proteins. The membrane binding sites for ankyrin include several integral membrane proteins, which are differentially expressed in different cells: the anion exchanger of intercalated cells of mammalian kidney, the sodium/potassium ATPase of kidney, and the voltage-dependent sodium channel of neurons. Ankyrin is present in many other cell types and it is likely that additional ankyrin-binding proteins will be identified. Each of the proteins that now are candidates for ankyrin binding proteins are ion channels or transporters and are localized in specialized cellular domains. The polarized localization of the ankyrin-associated membrane proteins is an essential aspect of their function at a physiological level. Spectrin and ankyrin thus exhibit an unsuspected diversity in protein linkages and have the potential for cell domain-specific interactions with a variety of membrane proteins. 相似文献
19.
As the determination of gene sequences and their function gains speed at the dawn of the third millennium, biomedical research efforts are oriented towards definition of the genetic and molecular expression patterns that may drive different disease. A major part of these efforts is addressed to the definition of inter-individual variations that are expected to become integral for treatment planning, in terms of efficacy and adverse effects of drugs. It is this thrust on genome-based 'rational therapeutics' that is hoped to progressively lead to the era of 'personalized medicine'. This approach uses the technological expertise from genomics and functional genomics to define, predict and monitor the nature of the response of an individual to drugs, and to rationally design newer drugs. In the present review we will conduct our readers through an understanding of the fundamentals of pharmacogenomics and of the technologies currently available that are advancing this relatively new science. Conversely, there are issues raised that concern how medical practice is preparing itself to implement new alternatives for therapeutical interventions and finally, how to respect patient confidentiality and rights. 相似文献
20.
Franklin A. Hays Zygy Roe-Zurz Min Li Libusha Kelly Franz Gruswitz Andrej Sali Robert M. Stroud 《Journal of structural and functional genomics》2009,10(1):9-16
Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane
proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering
high-quality, responsive non-redundant targets for crystallization. This “discovery-oriented” pipeline sidesteps two significant
bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins
that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column—a hallmark
of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker’s yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane
proteomes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Franklin A. Hays and Zygy Roe-Zurz have contributed equally to this work. 相似文献