Eukaryotic Gene Products Research Articles

In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases

A substantial fraction of eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) membrane. These polypeptides enter cotranslationally in the ER lumen, which contains resident molecular chaperones and folding factors that assist their maturation. Native proteins are released from the ER lumen and are transported through the secretory pathway to their final intra- or extracellular destination. Folding-defective polypeptides are exported across the ER membrane into the cytosol and destroyed. Cellular and organismal homeostasis relies on a balanced activity of the ER folding, quality control, and degradation machineries as shown by the dozens of human diseases related to defective maturation or disposal of individual polypeptides generated in the ER.

Efficient Restraints for Protein–Protein Docking by Comparison of Observed Amino Acid Substitution Patterns with those Predicted from Local Environment

The discovery that the functions of most eukaryotic gene products are mediated through multi-protein complexes makes the prediction of protein interactions one of the most important current challenges in structural biology. Rigid-body docking methods can generate a large number of alternative candidates, but it is difficult to discriminate the near-native interactions from the large number of false positives. Many different scoring functions have been developed for this purpose, but in most cases, experimental and biological information is still required for accurate predictions. We explore here the use of evolutionary restraints in evaluating rigid-body docking geometries. In order to identify potential interface residues we identify functional residues based on the comparison of observed amino acid substitutions with those predicted from local environment. The interface residues identified by this method are correctly located in 85% of the cases. These predicted interface residues are used to define distance restraints that help to score rigid-body docking solutions. We have developed the pyDockRST software, which uses the percentage of satisfied distance restraints, together with the electrostatics and desolvation binding energy, to identify correct docking orientations. This methodology dramatically improves the docking results when compared to the use of energy criteria alone, and is able to find the correct orientation within the top 20 docking solutions in 80% of the cases.

Domain architectural census of eukaryotic gene products containing O-protein phosphatases

Intricate molecular signalling within cellular environment is manifested through phosphorylation of proteins. Regulation of the phosphorylation state is executed through complex networking among kinases and their biochemical antagonists, the protein phosphatases. Protein dephosphorylation in eukaryotic systems is largely performed through four structurally distinct Ser/Thr and Tyr O-protein phosphatase superfamilies. 555 O-protein phosphatases, belonging to the four distinct families, could be identified using sensitive sequence search techniques across five eukaryotic model organisms (yeast, fly, worm, mouse and humans). These phosphatases could be grouped into 49 subfamilies associated with distinct domain architecture and discrete biochemical function. Only five of the architectures are shared across the five eukaryotic genomes. Interestingly, the number of occurrence of tyrosine phosphatases is correlated to the complexity of the genome. Analysis of domain architectures suggests amenability of the tyrosine phosphatases to occur in complex architectures unlike Ser/Thr phosphatases. Domain duplication and shuffling is shown as the customary mechanism for the evolution of the phosphatases. Several architectures are common between humans and other genomes, which are probably non-linearly inherited in humans or specifically lost in several others.

Two Proteins Homologous to the N- and C-terminal Domains of the Bacterial Glycosyltransferase Murg Are Required for the Second Step of Dolichyl-linked Oligosaccharide Synthesis in Saccharomyces cerevisiae

Two highly conserved eukaryotic gene products of unknown function showing homology to glycosyltransferases involved in the second steps of bacterial peptidoglycan (Murg) and capsular polysaccharide (Cps14f/Cps14g) biosynthesis have been identified in silico. The amino acid sequence of the eukaryotic protein that is homologous to the lipid acceptor- and membrane-associating N-terminal domain of Murg and the Cps14f beta4-galactosyltransferase enhancer protein is predicted to possess a cleavable signal peptide and transmembrane helices. The other eukaryotic protein is predicted to possess neither transmembrane regions nor a signal peptide but is homologous to the UDP-sugar binding C-terminal domain of Murg and the Cps14g beta4-galactosyltransferase. Both the eukaryotic proteins are encoded by essential genes in Saccharomyces cerevisiae, and down-regulation of either causes growth retardation, reduced N-glycosylation of carboxypeptidase Y, and accumulation of dolichyl-PP-GlcNAc. In vitro studies demonstrate that these proteins are required for transfer of [3H]GlcNAc from UDP-[3H]GlcNAc onto dolichyl-PP-GlcNAc. To conclude, two gene products showing homology to bacterial glycosyltransferases are required for the second step in dolichyl-PP-oligosaccharide biosynthesis.

Expression screening, protein purification and NMR analysis of human protein domains for structural genomics.

Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.

Annotation of glycoproteins in the SWISS-PROT database.

SWISS-PROT is a protein sequence database, which aims to be nonredundant, fully annotated and highly cross-referenced. Most eukaryotic gene products undergo co- and/or post-translational modifications, and these need to be included in the database in order to describe the mature protein. SWISS-PROT includes information on many types of different protein modifications. As glycosylation is the most common type of post-translational protein modification, we are currently placing an emphasis on annotation of protein glycosylation in SWISS-PROT. Information on the position of the sugar within the polypeptide chain, the reducing terminal linkage as well as additional information on biological function of the sugar is included in the database. In this paper we describe how we account for the different types of protein glycosylation, namely N-linked glycosylation, O-linked glycosylation, proteoglycans, C-linked glycosylation and the attachment of glycosyl-phosphatidylinosital anchors to proteins.

Annotation of glycoproteins in the SWISS-PROT database

SWISS-PROT is a protein sequence database, which aims to be nonredundant, fully annotated and highly cross-referenced. Most eukaryotic gene products undergo co- and/or post-translational modifications, and these need to be included in the database in order to describe the mature protein. SWISS-PROT includes information on many types of different protein modifications. As glycosylation is the most common type of post-translational protein modification, we are currently placing an emphasis on annotation of protein glycosylation in SWISS-PROT. Information on the position of the sugar within the polypeptide chain, the reducing terminal linkage as well as additional information on biological function of the sugar is included in the database. In this paper we describe how we account for the different types of protein glycosylation, namely N-linked glycosylation, O-linked glycosylation, proteoglycans, C-linked glycosylation and the attachment of glycosyl-phosphatidylinosital anchors to proteins.

The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon

The nucleotide sequences surrounding termination codons influence the efficiency of translational readthrough. In this report, we examined the sequence requirement for efficient readthrough of the UGA codon in the Sindbis virus genomic RNA which regulates production of the putative viral RNA polymerase, nsP4. The UGA codon and its neighboring nucleotide sequences were subcloned into a heterologous coding context, and readthrough efficiency was measured by cell-free translation of RNA transcripts in rabbit reticulocyte lysates. The CUA codon immediately downstream of the UGA codon was found to be sufficient for efficient translational readthrough. Further mutagenesis of residues in the CUA triplet demonstrated that mutations at the second or third residues following the UGA codon (U and A, respectively) had little effect on readthrough efficiency. In contrast, replacement of the cytidine residue immediately downstream of the UGA codon with any of the other three nucleotides (U, A, or G) dramatically reduced the readthrough efficiency from approximately 10% to less than 1%. These results show that a simple sequence context can allow efficient readthrough of UGA codons in a mammalian translation system. Interestingly, compilation studies of nucleotide sequences surrounding eukaryotic termination codons indicate a strong bias against cytidine residues immediately 3' to UGA termination codons. Taken together with our results, this bias may reflect a selective pressure for efficient translation termination for most eukaryotic gene products.

Streptomyces: a host for heterologous gene expression.

Streptomyces species offer many potential advantages as hosts for the expression and secretion of eukaryotic gene products. In this review we discuss the expression and localization signals that have been used to direct heterologous gene expression and the applications of these signals. Finally, we discuss future strategies aimed at increasing the capacity of this host for the high level production of biologically active eukaryotic gene products.

The pAS vector system and its application to heterologous gene expression in Escherichia coli.

There are numerous proteins of biological interest which cannot be obtained from natural sources in quantities sufficient for detailed biochemical and physical analysis. The limited bioavailability of these molecules has made it impossible to consider their potential utilization as either pharmacological agents and/or targets. One solution to this problem has been the development of recombinant vector systems which are designed to achieve efficient expression of cloned genes in a variety of biological systems. This paper will describe the development and application of a particular set of vectors which have been designed to achieve efficient expression of essentially any gene coding sequence in Escherichia coli. The system utilizes efficient phage-derived transcriptional and translational regulatory signals and provides a strong regulatable promoter, an antitermination mechanism to ensure efficient transcription across any gene insert, high stability and, when appropriate, efficient translation initiation information. In addition, a wide variety of host strains have been developed in order to help control, stabilize and maximize expression of various cloned genes. The system has now been used to express efficiently more than 75 different prokaryotic and eukaryotic gene products. The application of this system to the expression and characterization of several oncogene products will be described.

Regulation of a metallothionein-growth hormone hybrid gene in bovine papilloma virus.

We have constructed bovine papilloma virus recombinants carrying a hybrid gene in which human growth hormone structural sequences are fused to the promoter and presumptive control region of the mouse metallothionein-I gene. Mouse cells transformed with the recombinants synthesize metallothionein-growth hormone hybrid mRNA with the same 5' end as metallothionein mRNA. Hybrid mRNA is inducible by cadmium but not by dexamethasone, whereas the chromosomal metallothionein genes in the same cells are inducible by both agents. This indicates that heavy metals and glucocorticoids regulate the mouse metallothionein-I gene by independent mechanisms. Furthermore, because the transformed cells produce large quantities of human growth hormone polypeptide, it appears that bovine papilloma virus-metallothionein vectors may be useful for obtaining the efficient and regulatable expression of other eukaryotic gene products. We have constructed recombinants that allow the insertion of new structural sequences under the control of the metallothionein promoter.

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.