Frank Grosse

Production of spider silk proteins in tobacco and potato

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420–3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability—a property that is used to purify the spidroins by a simple and efficient procedure.

Composition and Hierarchical Organisation of a Spider Silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.

Domain structure of human nuclear DNA helicase II (RNA helicase A).

Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH2 terminus to the helicase core. Hence, an active helicase required part of the NH2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.

Centrosomal localization of DNA damage checkpoint proteins

During mitosis, the phosphatidylinositol‐3 (PI‐3) family‐related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA‐PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA‐PKcs, the catalytic subunit of DNA‐PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co‐immunoprecipitated with Ku70/86, the DNA binding subunits of DNA‐PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of α‐tubulin foci in the nucleus. Interestingly, γ‐tubulin was also present in the nucleus and co‐immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of γ‐tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of γ‐tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response. J. Cell. Biochem. 101: 451–465, 2007.

Interactions of human Cdc45 with the Mcm2-7 complex, the GINS complex, and DNA polymerases ! and " during S phase

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot‐like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle. The co‐localization of Cdc45 with active replication sites during S phase suggested that the human Cdc45 protein was part of the elongation complex. This view was corroborated by findings that Cdc45 interacted with the elongating DNA polymerases δ and ɛ, with Psf2, which is a component of the GINS complex as well as with Mcm5 and 7, subunits of the putative replicative DNA helicase complex. Hence, Cdc45 may play an important role in elongation of DNA replication by bridging the processive DNA polymerases δ and ɛ with the replicative helicase in the elongating machinery.

Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system.

DNA polymerase α-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim2) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strandin vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2·10−8 m and 1.3·10−8 m, respectively.

Initiation of eukaryotic DNA replication: Regulation and mechanisms

The accurate and timely duplication of the genome is a major task for eukaryotic cells. This process requires the cooperation of multiple factors to ensure the stability of the genetic information of each cell. Mutations, rearrangements, or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing failures, disease, or death. Because of the size of eukaryotic genomes, chromosomal duplication is accomplished in a multiparallel process. In human somatic cells between 10,000 and 100,000 parallel synthesis sites are present. This raises fundamental problems for eukaryotic cells to coordinate the start of DNA replication at each origin and to prevent replication of already duplicated DNA regions. Since these general phenomena were recognized in the middle of the 20th century the regulation and mechanisms of the initiation of eukaryotic DNA replication have been intensively investigated. These studies were carried out to find the essential factors involved in the process and to determine their functions during DNA replication. These studies gave rise to a model of the organization and the coordination of DNA replication within the eukaryotic cell. The elegant experiments carried out by Rao and Johnson (1970) (1), who fused cells in different phases of the cell cycle, showed that G1 cells are competent for replication of their chromosomes, but lack a specific diffusible factor required to activate their replicaton machinery and showed that G2 cells are incompetent for DNA replication. These findings suggested that eukaryotic cells exist in two states. In G1 phase, cells are competent to initiate DNA replication, which is subsequently triggered in S phase. After completion of S phase, cells in G2 are no longer able to initiate DNA replication and they require a transition through mitosis to reenable initiation of DNA replication to take place in the next S phase. The Xenopus cell-free replication system has proved a good model system in which to study DNA replication in vitro as well as the mechanism preventing rereplication within a single cell cycle (2). Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor, which binds to chromatin before the G1-S transition and which is displaced during replication (2, 3). These results were supported by genetic and biochemical experiments in Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) (4, 5). The investigation of cell division cycle mutants and the budding yeast origin of replication resulted in the concept of a prereplicative and a postreplicative complex of initiation proteins (6-9). These three individual concepts have recently started to merge and it has become obvious that initiation in eukaryotes is generally governed by the same ubiquitous mechanisms.

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2)

The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 μg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase α

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumorsuppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase α-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2×108 M−1. Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4×108 M−1, when RPA was coupled to the sensor chip via its smallest subunit, and 1×108 M−1, when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase α-primase complex (pol-prim) with a KA value of 1×1010 M−1. Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2×1010 M−1 and 5×109 M−1, respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1 – 320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.