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Palomo M.,Groningen Biomolecular science and Biotechnology Institute GBB | Pijning T.,Zernike Institute for Advanced Materials | Booiman T.,Groningen Biomolecular science and Biotechnology Institute GBB | Dobruchowska J.M.,Groningen Biomolecular science and Biotechnology Institute GBB | And 9 more authors.
Journal of Biological Chemistry | Year: 2011

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)7-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Rintisch C.,Max Delbrück Center for Molecular Medicine | Heinig M.,Max Delbrück Center for Molecular Medicine | Heinig M.,Max Planck Institute for Molecular Genetics | Bauerfeind A.,Max Delbrück Center for Molecular Medicine | And 18 more authors.
Genome Research | Year: 2014

Histone modifications are epigenetic marks that play fundamental roles in many biological processes including the control of chromatin-mediated regulation of gene expression. Little is known about interindividual variability of histone modification levels across the genome and to what extent they are influenced by genetic variation. We annotated the rat genome with histone modification maps, identified differences in histone trimethyl-lysine levels among strains, and described their underlying genetic basis at the genome-wide scale using ChIP-seq in heart and liver tissues in a panel of rat recombinant inbred and their progenitor strains. We identified extensive variation of histone methylation levels among individuals and mapped hundreds of underlying cis- and trans-acting loci throughout the genome that regulate histone methylation levels in an allele-specific manner. Interestingly, most histone methylation level variation was trans-linked and the most prominent QTL identified influenced H3K4me3 levels at 899 putative promoters throughout the genome in the heart. Cis- acting variation was enriched in binding sites of distinct transcription factors in heart and liver. The integrated analysis of DNA variation together with histone methylation and gene expression levels showed that histoneQTLs are an important predictor of gene expression and that a joint analysis significantly enhanced the prediction of gene expression traits (eQTLs). Our data suggest that genetic variation has a widespread impact on histone trimethylation marks that may help to uncover novel genotype-phenotype relationships. © 2014 Rintisch et al.


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2005

To study the relationships between societal visions on technology and food and industrial and academic genomics research.


Valk V.,Groningen Biomolecular science and Biotechnology Institute GBB | Lammerts van Bueren A.,Groningen Biomolecular science and Biotechnology Institute GBB | van der Kaaij R.M.,Groningen Biomolecular science and Biotechnology Institute GBB | Dijkhuizen L.,Groningen Biomolecular science and Biotechnology Institute GBB
FEBS Journal | Year: 2016

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2007

None


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2001

Multidrug resistance (MDR) is a major problem in the treatment of cancer. MDR is often induced by the overexpression of multidrug transport proteins of which the ATP-binding cassette (ABC) transmembrane proteins P-glycoprotein (Pgp) and the multidrug resistance associated protein 1 (MRP1) are the best characterized. Both proteins are located in the plasma membrane and are responsible for the active efflux of a broad spectrum of substrates (allocrites), including clinically important anti-cancer drugs. Studies to use the occurrence of MDR-related transporters such as Pgp and MRP1 as a tool in survival prognosis of cancer patients show variable outcome, and depend on the type of cancer. Since not all types of MDR can be correlated to the presence of known transport proteins, the identification and characterization of new transport proteins is a prerequisite in the development of cancer treatments. An interesting new member of the ABC transport proteins recently identified is the half-transporter MXR, also named BCRP or ABCP. MXR confers resistance to various important anti-cancer drugs, such as mitoxantrone, anthracyclines (doxorubicin), and the DNA topoisomerase I inhibitors CPT-11 and topotecan. Only two modulators have been reported to specifically inhibit MXR-mediated transport. Since modulators often show profound pharmacokinetic interaction with anti-cancer drugs, other MXR-specific modulators need to be identified. |Functional characterization of (transporter) proteins in mammalian cells is often hampered by the complexity of the regulation of gene expression, mRNA stability and protein lifetime. In this aspect, bacterial cells offer a much simpler system for characterization of activity and specificity. Equally important, bacterial expression allows the purification of larger quantities of protein that facilitates their characterization using purified proteoliposomes and eliminates possible cross-activities of other transporters. Furthermore, genetic manipulations can be exploited readily within the bacterial system.|We plan to overproduce MXR in Lactococcus lactis and purify the transport protein in large quantities. A first characterization of the transporter will be performed by transport assays using whole cells, purified membrane vesicles and proteoliposomes made of purified MXR protein and lipids. Transport and binding will be followed by the use of fluorescently or radioactively labeled allocrites. Modulators of transport activity will be identified by inhibition of these activities. As a second line of research, we will isolate the nucleotide-binding domain of MXR to test allocrites for stimulatory capability of the intrinsic ATPase activity. Results from our group with the nucleotide-binding domain of MRP1 show that its ATPase activity can be stimulated by MRP-specific substrates. Thirdly, we will characterize MXR on its ability to dimerize. Even though the homodimer seems to be functional, there are indications that heterodimer formation induces a higher resistance level. The putative partner for MXR in this heterodimer is unknown as yet. We plan to apply a bacterial two-hybrid system to investigate homodimer formation and to screen a mammalian cDNA bank for possible partners of MXR in heterodimer formation using MXR as bait.|In order to circumvent MXR-mediated MDR in cancer cells, the half-transporter needs to be characterized in more detail with respect to the allocrite and modulator specificity. Moreover, since the functional unit of the transporter appears to be a dimer, more information is needed concerning the dimerization and the identity of putative partner half-transporters. The characterization of MXR is a prerequisite to attack MDR in cancer cells in an efficient manner.


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2002

All cells are surrounded by one or more membranes that allow the maintenance of a unique internal environment. In addition, eukaryotic cells contain many subcompartments (endoplasmic reticulum, mitochondria, peroxisomes, chloroplasts, vacuoles), each surrounded by one or more membranes. Proteins are normally synthesized in the cytosol. This implicates that proteins that have their localization outside the cell or in one of these subcompartments, must be translocated across membranes in order to reach their final destination. Various translocation systems have been discovered for the transport of proteins across membranes and in general these systems are also involved in the insertion of membrane proteins into the membrane. In bacteria, the so-called Sec-system provides the general route for proteins to cross the cytoplasmic membrane and for membrane proteins to get inserted into this membrane. The Sec-system is composed of eight different proteins that form two complexes, a core complex that constitutes the protein-conducting channel (formed by SecY, SecE and SecG) and motor protein (SecA), and a complex containing SecD, SecF, YajC and YidC. Based on hypotheses postulated by different research groups, the function of the SecDFyajC-YidC complex may be diverse ranging from the regulation of channel formation to the active participation in insertion and assembly of membrane proteins. In this project, the role of the SecDFyajC-YidC complex within the Sec-system will be elucidated. For this purpose, an in vitro system will be established that will be used to test the various postulated functions of the complex and to analyze its interaction with the protein-conducting channel and the substrate proteins. The results are expected to provide a deeper understanding of how the two complexes of the Sec-system cooperate and how the same system can translocate proteins across and insert membrane proteins into the membrane.


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2007

ATP synthase is a membrane protein that uses energy from the transmembrane proton electrochemicai gradient to form ATP from ADP and phosphate [l]Th e enzyme is composed of two major domains, the transmembrane F0 domain and the membrane excluded F1 domain [Z] The current project focuses on the subunit c of the protein, which is an essentiai part of the F0 domain and participates in transmembrane proton conduction. Structural studies indicate that the subunit c arranges into ring structures comprising of 10 -12 subunits in different organisms However, littie is known about the type of interactions that affect the formation of c-rings in the ATPase complex In this project, we further investigate the stability and self aggregation propensity of the c subunit, Molecular dynamics simulations wil1 be performed using a coarse-grain force field [3] t0 define the protein and its membrane environment Simulations wil1 be carried out from a single monomer as weli as the pre-assembied modelled ring structure of the c subunit The stability and dynamics of these systems wil1 be probed and the results wil1 be compared to atomistic simulations [4] Further, simulations wil1 be performed on multiple copies of the monomer to probe the association of these peptides The free energy profile of association of two peptides wil1 be caicuiated. Preliminary results indicate that the subunit has a tendency to assocate in the membrane into dimers and trimers. However, to investigate the more complex decarner interactions, longer time scales and larger length scales need to be investigated


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2008

None


PubMed | Groningen Biomolecular science and Biotechnology Institute GBB
Type: Journal Article | Journal: The FEBS journal | Year: 2016

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 -amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on -glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain -amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of -amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in -amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

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