Design of multivalent complexes using the barnase*barstar module

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.     

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.     

Production of combinatorial libraries of fused genes by sequential transposition reactions

The use of in vivo and in vitro transposition reactions to perform non-combinatorial manipulation of DNAs in molecular biology is widespread. In this work we describe a technique that utilizes two sequential, directed transposition reactions in order to carry out combinatorial DNA manipulations. The methodology relies on the use of two different mutant Tn5 transposase proteins that have different transposon end recognition specificities. We demonstrate that the technique can be used to create large libraries of random fusions between two genes. These transpositional fusions are defined by insertion of a 32 bp linker sequence. We applied the technique to a model system, chloramphenicol acetyl transferase, to create functional fusions from N- and C-terminally truncated, non-functional genes. Comparative structural analysis suggests that both sides of the linker are inserted into disordered regions in functional proteins.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

Weird fingers: Functional analysis of WIP domain proteins

WIP proteins form a plant specific subfamily of C2H2 zinc finger (ZF) proteins. In this study, we functionally characterized the WIP domain, which consists of four ZF motifs, and discuss molecular functions for WIP proteins. Mutations in each of the ZFs lead to loss of function of the TT1/WIP1 protein in Arabiopsis thaliana. SV40 type nuclear localisation signals were detected in two of the ZFs and functionally characterized using GFP fusions as well as new mutant alleles identified by TILLING. Promoter swap experiments showed that selected WIP proteins are partially able to take over TT1 function. Activity of the AtBAN promoter, a potential TT1 target, could be increased by the addition of TT1 to the TT2-TT8-TTG1 regulatory complex.     

The molecular signature of oncofusion proteins in acute myeloid leukemia

Acute myeloid leukemia (AML) associated translocations often cause gene fusions that encode oncofusion proteins. Although many of the breakpoints involved in chromosomal translocations have been cloned, in most cases the role of the chimeric proteins in tumorigenesis is not elucidated. Here we will discuss the fusion proteins of the 4 most common translocations associated with AML as well as the common molecular mechanisms that these four and other fusion proteins utilize to transform progenitor cells. Intriguingly, although the individual partners within the fusion proteins represent a wide variety of cellular functions, at the molecular level many commodities can be found.     

Action of a Transposable Element in Coding Sequence Fusions

The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.     

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.     

Reconstitution of the Kaposi's sarcoma-associated herpesvirus nuclear egress complex and formation of nuclear membrane vesicles by coexpression of ORF67 and ORF69 gene products

The Kaposi's sarcoma-associated herpesvirus nuclear egress complex is composed of two proteins, ORF67 and ORF69. In this study, we have recapitulated the KSHV complex by coexpression of these two proteins in insect cells using expression from recombinant baculoviruses. The proteins form a complex at the nuclear membrane as judged by live-cell analysis of protein fusions tagged with green fluorescent protein (GFP) and mCherry. Ultrastructural analysis of infected cells showed that ORF67 expression results in reduplication of the nuclear membrane. When the two proteins are expressed together, numerous virion-size nuclear membrane-derived vesicles were evident at the nuclear margins.     

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

Oxidative stress,mitochondrial bioenergetics,and cardiolipin in aging

Aging is a natural, complex, and multifactorial biological process associated with impairment of bioenergetic function, increased oxidative stress, attenuated ability to respond to stresses, and increased risk of contracting age-associated diseases. Oxidative stress is widely thought to underpin many aging processes. The mitochondrion, the powerhouse of the cell, is considered the most important cellular organelle to contribute to the aging process, mainly through respiratory chain dysfunction and formation of reactive oxygen species, leading to damage to mitochondrial proteins, lipids, and mitochondrial DNA. Cardiolipin, a phospholipid located at the level of the inner mitochondrial membrane, is known to be intimately involved in several mitochondrial bioenergetic processes as well as mitochondrial-dependent steps in apoptosis and mitochondrial membrane stability and dynamics. Alterations to cardiolipin structure, content, and acyl chain composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions and aging. In this review, we discuss several aspects of mitochondrial bioenergetic alterations in aging and the role played by reactive oxygen species and cardiolipin in these alterations.     

The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs(-/-) cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.     

Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the ³⁵S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.     

Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome

Plants with transgenic plastid (chloroplast) genomes represent a promising production platform in molecular farming, mainly because of the plastids' potential to accumulate foreign proteins to very high levels and the increased biosafety conferred by the maternal mode of plastid inheritance. Although some transgenes can be expressed to extraordinarily high levels, the expression of others has been unsuccessful. Lack of detectable transgene expression is usually attributable to either RNA instability or protein instability. Here, we have investigated the possibilities to improve the production of a pharmaceutical protein that is difficult to express in chloroplasts: the HIV-1 fusion inhibitor cyanovirin-N (CV-N). Testing various N-terminal and C-terminal fusions to peptide sequences from two proteins known to accumulate to high levels in transgenic plastids (GFP and the protein antibiotic PlyGBS), we show that both low mRNA stability and low protein stability contribute to the lack of detectable CV-N expression in chloroplasts. Both problems can be alleviated by N-terminal fusions to the CV-N coding region, thus highlighting a suitable strategy for optimization of plastid transgene expression.     

Evolution of Mitochondria Reconstructed from the Energy Metabolism of Living Bacteria

The ancestors of mitochondria, or proto-mitochondria, played a crucial role in the evolution of eukaryotic cells and derived from symbiotic α-proteobacteria which merged with other microorganisms - the basis of the widely accepted endosymbiotic theory. However, the identity and relatives of proto-mitochondria remain elusive. Here we show that methylotrophic α-proteobacteria could be the closest living models for mitochondrial ancestors. We reached this conclusion after reconstructing the possible evolutionary pathways of the bioenergy systems of proto-mitochondria with a genomic survey of extant α-proteobacteria. Results obtained with complementary molecular and genetic analyses of diverse bioenergetic proteins converge in indicating the pathway stemming from methylotrophic bacteria as the most probable route of mitochondrial evolution. Contrary to other α-proteobacteria, methylotrophs show transition forms for the bioenergetic systems analysed. Our approach of focusing on these bioenergetic systems overcomes the phylogenetic impasse that has previously complicated the search for mitochondrial ancestors. Moreover, our results provide a new perspective for experimentally re-evolving mitochondria from extant bacteria and in the future produce synthetic mitochondria.     

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants

A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of β-glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.     

SNARE蛋白调控细胞自噬的分子机制

细胞自噬是细胞在面对内外部环境压力的情况下, 为了自身的稳定而采取的一种降解内部及外来入侵物质的机制。SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptors)假说指出SNARE蛋白在细胞物质运输以及特异性膜融合过程中具有重要作用, 揭示了细胞正常生理活动有序进行的分子机制。由于细胞自噬涉及从自噬体的形成到自噬体溶酶体的融合等诸多膜融合的过程, 因此, 文章对近年来SNARE蛋白在调控细胞自噬过程的研究进展进行了综述。