Structural insights of proteins in sub-cellular compartments: In-mitochondria NMR

Many eukaryotic proteins exert their physiological function in specific cellular compartments. Proteins of the inter-membrane space (IMS) of mitochondria, for example, are synthesized in the cytoplasm and translocate to the IMS, where they are further processed to their mature form. In-cell Nuclear Magnetic Resonance (NMR) has proven to be an ideal approach to investigate eukaryotic proteins at the atomic level, inside the cytoplasm. Here we show that proteins inside intact mitochondria isolated from human cells can be structurally characterized by NMR (in-mitochondria NMR). By this approach, we characterized the folding and maturation state of two human proteins in the IMS, SOD1 and Mia40. Both observed proteins were in the folded state. Mia40 was in the oxidized, functional state, while SOD1 disulfide bond formation was promoted by increasing the level of the SOD1 chaperone, CCS, in the IMS.     

Functional expression of eukaryotic membrane proteins in Lactococcus lactis

The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin-inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N-terminal regions of the transporters. Expression levels were improved >10-fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high-throughput functional characterization of eukaryotic membrane proteins and structural genomics.     

Replication licensing--defining the proliferative state?

The proliferation of eukaryotic cells is a highly regulated process that depends on the precise duplication of chromosomal DNA in each cell cycle. Regulation of the replication licensing system, which promotes the assembly of complexes of proteins termed Mcm2-7 onto replication origins, is responsible for preventing re-replication of DNA in a single cell cycle. Recent work has shown how the licensing system is directly controlled by cyclin-dependent kinases (CDKs). Repression of origin licensing is emerging as a ubiquitous route by which the proliferative capacity of cells is lowered, and Mcm2-Mcm7 proteins show promise as diagnostic markers of early cancer stages. These results have prompted us to propose a functional distinction between the proliferative state and the non-proliferative state (including G0) depending on whether origins are licensed.     

Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.     

Prenylation: From bacteria to eukaryotes

For their protection from host cell immune defense, intracellular pathogens of eukaryotic cells developed a variety of mechanisms, including secretion systems III and IV which can inject bacterial effectors directly into eukaryotic cells. These effectors may function inside the host cell and may be posttranslationally modified by host cell machinery. Recently, prenylation was added to the list of possible posttranslational modifications of bacterial proteins. In this work we describe the current state of the knowledge about the prenylation of eukaryotic and prokaryotic proteins and prenylation inhibitors. The bioinformatics analyses suggest the possibility of prenylation for a number of Francisella genus proteins.     

Messenger ribonucleoproteins (informosomes) and RNA-binding proteins

Messenger ribonucleoproteins, first discovered in 1964 in our laboratory as free mRNA-containing particles of fish embryo cytoplasm and designated as informosomes, proved to have a universal occurrence in eukaryotic cells. Messenger ribonucleoproteins of different intracellular localization such as free cytoplasmic non-translatable informosomes, translatable messenger ribonucleoproteins in polyribosomes and nuclear pre-mRNA-containing particles are characterized by a number of features common for all of them. However, the transport from the nucleus into the cytoplasm as well as the transition from the free non-translatable state into the polyribosome-bound translatable state are accompanied by essential changes in the protein moiety of the particles. The existance of free RNA-binding proteins in eukaryotic cells has also been shown. These proteins seem to represent a pool for the formation of messenger ribonucleoproteins (informosomes).It has recently been demonstrated that the eukaryotic translation factors and, in particular, both the elongation factors and some initiation factors are among the cytoplasmic RNA-binding proteis. It is suggested that the mRNA in eukaryotic cells at different stages of its life time carries on itself the proteins which are required for its own biogenesis, processing and transport (nuclear informosomes), for its existence in a temporarily inactive state (free cytoplasmic informosomes) and for its functioning as a template (polyribosomal informosomes):omnia mea mecum porto.     

Haspin-like proteins: a new family of evolutionarily conserved putative eukaryotic protein kinases

Haspin (haploid germ cell-specific nuclear protein kinase) is reported to be a serine/threonine kinase that may play a role in cell-cycle cessation and differentiation of haploid germ cells. In addition, Haspin mRNA can be detected in diploid cell lines and tissues. Here, Haspin-like proteins are identified in several major eukaryotic phyla-including yeasts, plants, flies, fish, and mammals-and an extended group in Caenorhabditis elegans. The Haspin-like proteins have a complete but divergent eukaryotic protein kinase domain sequence. Although clearly related to one another and to other eukaryotic protein kinases, the Haspin-related proteins lack conservation of a subset of residues that are almost invariant in known kinases and possess distinctive inserted regions. In fact, phylogenetic analysis indicates that the Haspin-like proteins form a novel eukaryotic protein kinase family distinct from those previously defined. The identification of related proteins in model organisms provides some initial insight into their functional properties and will provide new experimental avenues by which to determine the function of the Haspin proteins in mammalian cells.     

Purification and identification of novel Rab effectors using affinity chromatography

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.     

Bacterial ancestry of actin and tubulin

The structural and functional resemblance between the bacterial cell-division protein FtsZ and eukaryotic tubulin was the first indication that the eukaryotic cytoskeleton may have a prokaryotic origin. The bacterial ancestry is made even more obvious by the findings that the bacterial cell-shape-determining proteins Mreb and Mbl form large spirals inside non-spherical cells, and that MreB polymerises in vitro into protofilaments very similar to actin. Recent advances in research on two proteins involved in prokaryotic cytokinesis and cell shape determination that have similar properties to the key components of the eukaryotic cytoskeleton are discussed.     

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells

G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.     

Functional reconstitution of a tunable E3-dependent sumoylation pathway in Escherichia coli

SUMO (small ubiquitin-related modifier) is a reversible post-translational protein modifier that alters the localization, activity, or stability of proteins to which it is attached. Many enzymes participate in regulated SUMO-conjugation and SUMO-deconjugation pathways. Hundreds of SUMO targets are currently known, with the majority being nuclear proteins. However, the dynamic and reversible nature of this modification and the large number of natively sumoylated proteins in eukaryotic proteomes makes molecular dissection of sumoylation in eukaryotic cells challenging. Here, we have reconstituted a complete mammalian SUMO-conjugation cascade in Escherichia coli cells that involves a functional SUMO E3 ligase, which effectively biases the sumoylation of both native and engineered substrate proteins. Our sumo-engineered E. coli cells have several advantages including efficient protein conjugation and physiologically relevant sumoylation patterns. Overall, this system provides a rapid and controllable platform for studying the enzymology of the entire sumoylation cascade directly in living cells.     

Cell-free expression systems for eukaryotic protein production

Following the success of genome sequencing projects, attention has now turned to studies of the structure and function of proteins. Although cell-based expression systems for protein production have been widely used, they have certain limitations in terms of the quality and quantity of the proteins produced and for high-throughput production. Many of these limitations can be circumvented by the use of cell-free translation systems. Among such systems, the wheat germ based system is of special interest for its eukaryotic nature; it has the significant advantage of producing eukaryotic multidomain proteins in a folded state. Several advances in the use of cell-free expression systems have been made in the past few years and successful applications of these systems to produce proteins for functional and structural biology studies have been reported.     

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.     

Probes for studying cholesterol binding and cell biology

Cholesterol is a multifunctional lipid in eukaryotic cells. It regulates the physical state of the phospholipid bilayer, is crucially involved in the formation of membrane microdomains, affects the activity of many membrane proteins, and is the precursor for steroid hormones and bile acids. Thus, cholesterol plays a profound role in the physiology and pathophysiology of eukaryotic cells. The cholesterol molecule has achieved evolutionary perfection to fulfill its different functions in membrane organization. Here, we review basic approaches to explore the interaction of cholesterol with proteins, with a particular focus on the high diversity of fluorescent and photoreactive cholesterol probes available today.     

Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: a lattice study

MOTIVATION: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. RESULTS: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.     

Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins

To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution.     

Chromatin as a eukaryotic template of genetic information

In eukaryotes, chromatin is essential for heredity. Chromatin architecture is sometimes "epistatic" over the DNA and imparts a different heritable state to the same DNA sequence or the same functional state to unrelated DNA sequences. This has been documented recently in a wide variety of studies focused on regulation of the yeast mating type, the function of Polycomb and trithorax group proteins, the specification of eukaryotic centromeres and neocentromeres, and genomic imprinting.     

Functional properties of a recombinant bacterial DING protein: comparison with a homologous human protein

DING proteins are highly-conserved proteins with poorly-defined cell-signalling roles in mammals. Conserved homologues are also commonplace in plants, though not as yet functionally characterized. Poor availability of the proteins, and a lack of genetic structure, hamper progress in elucidating the roles of these eukaryotic DING proteins, but highly-homologous hypothetical DING proteins have recently been identified in Pseudomonas genomes. We have cloned and expressed a DING protein from P. fluorescens SWB25 in Escherichia coli. The recombinant protein, and its natural human homologue, act as phosphate-binding proteins, as predicted by structural homologies with other bacterial proteins. The recombinant protein also displays other functional similarities with mammalian DING proteins, in that, like the human version, it acts as a mitogen for cultured human cells, and can bind cotinine, known to be a binding ligand for a rat neuronal DING protein.