Center for Integrated Protein Science Munich

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

Hydrogen evolution from photocatalytic reduction of water holds promise as a sustainable source of carbon-free energy. Covalent organic frameworks (COFs) present an interesting new class of photoactive materials, which combine three key features relevant to the photocatalytic process, namely crystallinity, porosity and tunability. Here we synthesize a series of water- and photostable 2D azine-linked COFs from hydrazine and triphenylarene aldehydes with varying number of nitrogen atoms. The electronic and steric variations in the precursors are transferred to the resulting frameworks, thus leading to a progressively enhanced light-induced hydrogen evolution with increasing nitrogen content in the frameworks. Our results demonstrate that by the rational design of COFs on a molecular level, it is possible to precisely adjust their structural and optoelectronic properties, thus resulting in enhanced photocatalytic activities. This is expected to spur further interest in these photofunctional frameworks where rational supramolecular engineering may lead to new material applications.

The neurodegeneration observed in Alzheimer's disease has been associated with synaptic dismantling and progressive decrease in neuronal activity. We tested this hypothesis in vivo by using two-photon Ca2+ imaging in a mouse model of Alzheimer's disease. Although a decrease in neuronal activity was seen in 29% of layer 2/3 cortical neurons, 21% of neurons displayed an unexpected increase in the frequency of spontaneous Ca2+ transients. These "hyperactive" neurons were found exclusively near the plaques of amyloid beta-depositing mice. The hyperactivity appeared to be due to a relative decrease in synaptic inhibition. Thus, we suggest that a redistribution of synaptic drive between silent and hyperactive neurons, rather than an overall decrease in synaptic activity, provides a mechanism for the disturbed cortical function in Alzheimer's disease.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.

Chromosome territories (CTs) constitute a major feature of nuclear architecture. In a brief statement, the possible contribution of nuclear architecture studies to the field of epigenomics is considered, followed by a historical account of the CT concept and the final compelling experimental evidence of a territorial organization of chromosomes in all eukaryotes studied to date. Present knowledge of nonrandom CT arrangements, of the internal CT architecture, and of structural interactions with other CTs is provided as well as the dynamics of CT arrangements during cell cycle and postmitotic terminal differentiation. The article concludes with a discussion of open questions and new experimental strategies to answer them.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation channels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart and nervous system. The current produced by HCN channels has been known as I(h) (or I(f) or I(q)). I(h) has also been designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemaker cells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidence that I(h) is also involved in a number of basic physiological processes that are not directly associated with rhythmicity. Examples for these non-pacemaking functions of I(h) are the determination of the resting membrane potential, dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into the structure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCN channel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channel types arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of the impact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCN channels as drug targets.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

5-Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5-formylcytosine (fC), 5-carboxylcytosine (caC), and 5-hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.

Genome-, transcriptome- and proteome-wide measurements provide insights into how biological systems are regulated. However, fundamental aspects relating to which human proteins exist, where they are expressed and in which quantities are not fully understood. Therefore, we generated a quantitative proteome and transcriptome abundance atlas of 29 paired healthy human tissues from the Human Protein Atlas project representing human genes by 18,072 transcripts and 13,640 proteins including 37 without prior protein-level evidence. The analysis revealed that hundreds of proteins, particularly in testis, could not be detected even for highly expressed mRNAs, that few proteins show tissue-specific expression, that strong differences between mRNA and protein quantities within and across tissues exist and that protein expression is often more stable across tissues than that of transcripts. Only 238 of 9,848 amino acid variants found by exome sequencing could be confidently detected at the protein level showing that proteogenomics remains challenging, needs better computational methods and requires rigorous validation. Many uses of this resource can be envisaged including the study of gene/protein expression regulation and biomarker specificity evaluation.

Light is a fascinating phenomenon that ties together physics, chemistry, and biology. It is unmatched in its ability to confer information with temporal and spatial precision and has been used to map objects on the scale of tens of nanometers (10(-8) m) to light years (10(16) m). This information, gathered through super-resolution microscopes or space-based telescopes, is ultimately funneled through the human visual system, which is a miracle in itself. It allows us to see the Andromeda galaxy at night, an object that is 2.5 million light years away and very dim, and ski the next day in bright sunlight at an intensity that is 12 orders of magnitude higher. Human vision is only one of many photoreceptive systems that have evolved on earth and are found in all kingdoms of life. These systems rely on molecular photoswitches, such as retinal or tetrapyrrols, which undergo transient bond isomerizations or bond formations upon irradiation. The set of chromophores that have been employed in Nature for this purpose is surprisingly small. Nevertheless, they control a wide variety of biological functions, which have recently been significantly increased through the rapid development of optogenetics. Optogenetics originated as an effort to control neural function with genetically encoded photoreceptors that use abundant chromophores, in particular retinal. It now covers a variety of cellular functions other than excitability and has revolutionized the control of biological pathways in neuroscience and beyond. Chemistry has provided a large repertoire of synthetic photoswitches with highly tunable properties. Like their natural counterparts, these chromophores can be attached to proteins to effectively put them under optical control. This approach has enabled a new type of synthetic photobiology that has gone under various names to distinguish it from optogenetics. We now call it photopharmacology. Here we trace our involvement in this field, starting with the first light-sensitive potassium channel (SPARK) and concluding with our most recent work on photoswitchable fatty acids. Instead of simply providing a historical account of our efforts, we discuss the design criteria that guided our choice of molecules and receptors. As such, we hope to provide a roadmap to success in photopharmacology and make a case as to why synthetic photoswitches, properly designed and made available through well-planned and efficient syntheses, should have a bright future in biology and medicine.

Alzheimer's disease (AD) is characterized by a progressive dysfunction of central neurons. Recent experimental evidence indicates that in the cortex, in addition to the silencing of a fraction of neurons, other neurons are hyperactive in amyloid-β (Aβ) plaque-enriched regions. However, it has remained unknown what comes first, neuronal silencing or hyperactivity, and what mechanisms might underlie the primary neuronal dysfunction. Here we examine the activity patterns of hippocampal CA1 neurons in a mouse model of AD in vivo using two-photon Ca(2+) imaging. We found that neuronal activity in the plaque-bearing CA1 region of older mice is profoundly altered. There was a marked increase in the fractions of both silent and hyperactive neurons, as previously also found in the cortex. Remarkably, in the hippocampus of young mice, we observed a selective increase in hyperactive neurons already before the formation of plaques, suggesting that soluble species of Aβ may underlie this impairment. Indeed, we found that acute treatment with the γ-secretase inhibitor LY-411575 reduces soluble Aβ levels and rescues the neuronal dysfunction. Furthermore, we demonstrate that direct application of soluble Aβ can induce neuronal hyperactivity in wild-type mice. Thus, our study identifies hippocampal hyperactivity as a very early functional impairment in AD transgenic mice and provides direct evidence that soluble Aβ is crucial for hippocampal hyperactivity.

Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein. Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein. After the identification of most components of the cell, further insights into their regulation and function require information on their abundance, localization, and dynamic interactions. Green fluorescent proteins (GFPs) 1The abbreviations used are: GFP, green fluorescent protein; GBP, GFP-binding protein; ChIP, chromatin immunoprecipitation; VHH, variable domain of heavy chain antibody; HEK, human embryonic kidney; IgG, immunoglobulin G; YFP, enhanced yellow fluorescent protein; CFP, enhanced cyan fluorescent protein; DsRed, Discosoma genus red fluorescent protein; mRFP, monomeric red fluorescent protein; PCNA, proliferating cell nuclear antigen; H2B, histone H2B; Dnmt1, DNA methyltransferase I; PBD, PCNA binding domain; HMGA1a, high mobility group protein A1a; Igf, insulin-like growth factor; PML, promyelocytic leukemia protein. 1The abbreviations used are: GFP, green fluorescent protein; GBP, GFP-binding protein; ChIP, chromatin immunoprecipitation; VHH, variable domain of heavy chain antibody; HEK, human embryonic kidney; IgG, immunoglobulin G; YFP, enhanced yellow fluorescent protein; CFP, enhanced cyan fluorescent protein; DsRed, Discosoma genus red fluorescent protein; mRFP, monomeric red fluorescent protein; PCNA, proliferating cell nuclear antigen; H2B, histone H2B; Dnmt1, DNA methyltransferase I; PBD, PCNA binding domain; HMGA1a, high mobility group protein A1a; Igf, insulin-like growth factor; PML, promyelocytic leukemia protein. and spectral variants thereof became popular tools to determine protein localization and, in combination with fluorescence photobleaching techniques, provided unique information on protein dynamics in living cells (1Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5451) Google Scholar, 2Misteli T. Spector D.L. Applications of the green fluorescent protein in cell biology and biotechnology.Nat. Biotechnol. 1997; 15: 961-964Crossref PubMed Scopus (307) Google Scholar, 3Tsien R.Y. The green fluorescent protein.Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar, 4van Roessel P. Brand A.H. Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins.Nat. Cell Biol. 2002; 4: E15-E20Crossref PubMed Scopus (211) Google Scholar). Necessary additional information on DNA binding, enzymatic activity, and complex formation can be obtained with various methods including chromatin immunoprecipitation (ChIP) and affinity and of 2002; PubMed Scopus Google Scholar, of chromatin in vivo 1997; PubMed Scopus Google Scholar). are the of specific are the protein of to specific protein including for PubMed Scopus Google Scholar, of protein from and biochemical to Biotechnol. PubMed Scopus Google Scholar). GFP, the most widely used in cell is used for biochemical various and of fluorescent protein to study nuclear of the in living Biol. 15: PubMed Scopus Google Scholar, proteins as 4: PubMed Scopus Google Scholar). This be in to and as as heavy and with to are variable single domain also to as VHH, derived from heavy chain of T. G. of PubMed Scopus Google Scholar). the with a mass of are and and can be produced in the Biol. PubMed Scopus Google Scholar, domain Biotechnol. Google Scholar). used for various P. in the of the human PubMed Scopus Google Scholar, of enzyme function in Biotechnol. PubMed Scopus Google Scholar, P. P. of specific for the domain in PubMed Scopus Google we a of a 13-kDa GFP binding fragment derived from a llama single chain and in cells with fluorescent PubMed Scopus Google Scholar). This GFP-binding protein (GBP) a and can easily be produced in We immobilized the GBP to a enables a fast and efficient isolation of GFP fusion proteins and their interacting factors for biochemical and Moreover we the GFP-binding protein can be fused with proteins to ectopically recruit GFP fusion proteins and interacting factors in living the localization and binding dynamics of fluorescent fusion proteins be with biochemical on interacting enzymatic activity, and DNA binding a for fast and efficient of GFP fusion proteins we used a 13-kDa GBP the domain of a heavy chain in GFP and in cells with fluorescent PubMed Scopus Google Scholar). The GBP fused with a produced in and a single the GBP can easily be produced and as a a functional we the binding of the GBP to of proteins and for the GBP and into a complex with a mass of We the of GBP to from cell and with and GBP and to protein of cells expressing proteins and After with GBP protein and the GBP additional to the and heavy of the as as proteins be in immunoprecipitations with the and The of GFP in to the GBP is efficient and and of fluorescent immunoprecipitation of of GBP with and of cells to immunoprecipitation with GBP, of and and GFP, heavy and of the and the GBP are with the GFP-nanotrap. protein on a GBP coupled to and proteins of GFP fusion proteins with the GFP-nanotrap. of cells are and GFP fusion proteins with the GFP-nanotrap. of GFP fusion proteins and interacting cells a of with the red fluorescent of PCNA cell of cells in are cells and to immunoprecipitation with the GFP-nanotrap. GBP can be immobilized and with protein to of the GBP and to with analyses we coupled GBP to a GFP-binding This GFP-nanotrap a fast of GFP from cell a single protein protein Moreover the a of the as of GFP from the the of GBP we immunoprecipitation with a of fluorescent proteins. GFP GBP the yellow of mRFP, further the binding of GBP we and we the of the binding from to and GBP high we and of GFP the and binding of we the of GFP in the range from to the to of GFP a fast and efficient to from the GFP-nanotrap GBP also GFP fusion proteins we from M. M. G. G. dynamics cell and a fusion Biol. 1997; PubMed Scopus Google G. and a in Cell Biol. PubMed Scopus Google P. T. of DNA in living Cell Biol. PubMed Scopus Google and T. fusion protein enables of dynamics in living Biol. 1998; PubMed Scopus Google Scholar). expressing the fusion proteins to determine the localization of the GFP fusion protein cells and GFP fusion proteins with the GFP-nanotrap and lower The GBP GFP fusion proteins from and of the most of the GBP is the to localization, and complex formation of specific proteins and We a of the PCNA domain chromatin of and PubMed Scopus Google Scholar). with the fluorescent with the red fluorescent PCNA We from living cells with biochemical and to immunoprecipitation with the GFP-nanotrap. with with lower The the of the in of DNA methyltransferase activity in living PubMed Scopus Google Scholar). we also PCNA with with GBP can also interacting proteins. the GFP-nanotrap is suitable to interacting factors and cell of and specific binding we the GFP-nanotrap to interacting factors mass in cell is the fluorescent fusion proteins are and biochemical to their fast of enzymatic activity we fluorescent fusion of the Dnmt1, are in cell we the and After of the fluorescent in of cells proteins from cell with the GFP-nanotrap and for enzymatic measurements. a specific DNA methyltransferase activity to of lower The with the the DNA methyltransferase of cells the GFP-nanotrap is a efficient for and biochemical of fluorescent fusion fusion proteins their enzymatic activity with the GFP-nanotrap. of from cells the Dnmt1, protein one with the GFP-nanotrap and DNA methyltransferase activity of GFP fusion proteins and the GFP-nanotrap the to function in chromatin immunoprecipitations we a cell expressing a GFP fusion of the high mobility group protein and We the of the and protein are to M. components chromatin Biol. PubMed Google Scholar, nuclear and high mobility group proteins the of the insulin-like growth 1997; PubMed Scopus Google Scholar, G. M. of gene function embryonic cell PubMed Scopus Google Scholar, M. G. of the and in and PubMed Scopus Google Scholar). of to immunoprecipitation with the GFP-nanotrap and with the GFP-nanotrap a of This binding of the specific with The cells expressing specific of in with the the high and of the with the GFP-nanotrap. of cells to immunoprecipitation of of and of DNA of the and and obtained for the and are in the obtained in as the obtained with the GFP-nanotrap and in vitro of the GFP-binding protein. a we in vivo and the of the GFP-binding protein a the cell to a of the nuclear We fused the GBP with to a cellular the nuclear GBP and the in vitro biochemical with binding in living cells we cells with GFP, and and the of GFP and with to the GFP-nanotrap the nuclear GFP the nuclear a the cell of GFP, YFP, and from the fluorescent proteins the GBP we single to We the of to binding to the to a binding This the high and of the GBP in vitro and in vivo allowing of single we the GFP-nanotrap can be used to factors and cellular a we the The is in nuclear a in regulation of gene DNA and P. and of Biol. PubMed Scopus Google Scholar, the promyelocytic leukemia protein for nuclear Biol. 2002; PubMed Scopus Google Scholar). We of in cells are the cells are the green fluorescence of the is for protein and cell is for the formation of Google fusion protein and with a specific expression of green fluorescent the nuclear and the of from the nuclear the of the GFP-nanotrap to recruit GFP fusion proteins also interacting of the in cells expressing GFP, CFP, and GFP is the nuclear is of GFP, CFP, and are in The of GFP The of green fluorescent Biotechnol. PubMed Scopus Google is and the is as a protein of cells expressing GFP, CFP, and to with the GFP-nanotrap. and and and with manipulation of nuclear structures with the GFP-nanotrap. cells in combination with are are on the is into with antibody. of the with to a of from the nuclear the of in the nuclear with of the is the of biochemical, and cell This in the protein are used for we a GBP as a and versatile for biochemical analyses of GFP fusion proteins and functional in The and GBP the and the GBP can be produced in bacteria in and with and can easily be coupled to the high affinity of single chain GBP allows to fluorescent fusion proteins and factors from cellular size of binding and heavy and of and with of the GFP-nanotrap further with cells expressing a DNA binding gene with the GFP-nanotrap we a binding of the with versatile GFP-nanotrap with the of fluorescent fusion proteins enables a fast and of the localization and mobility of fluorescent fusion proteins with their enzymatic activity, interacting and DNA binding cell biology and with the GFP-nanotrap can also be used for functional in We GBP can be fused with cellular proteins to a GFP-nanotrap a to ectopically recruit GFP fusion proteins and their factors from their one we the GBP the nuclear to capture nuclear are GFP fusion protein can be with cellular The of GBP as a in living cells of of functional to and cellular processes and After the identification of most components of the cell, further insights into their regulation and function require information on their abundance, localization, and dynamic interactions. Green fluorescent proteins (GFPs) 1The abbreviations used are: GFP, green fluorescent protein; GBP, GFP-binding protein; ChIP, chromatin immunoprecipitation; VHH, variable domain of heavy chain antibody; HEK, human embryonic kidney; IgG, immunoglobulin G; YFP, enhanced yellow fluorescent protein; CFP, enhanced cyan fluorescent protein; DsRed, Discosoma genus red fluorescent protein; mRFP, monomeric red fluorescent protein; PCNA, proliferating cell nuclear antigen; H2B, histone H2B; Dnmt1, DNA methyltransferase I; PBD, PCNA binding domain; HMGA1a, high mobility group protein A1a; Igf, insulin-like growth factor; PML, promyelocytic leukemia protein. 1The abbreviations used are: GFP, green fluorescent protein; GBP, GFP-binding protein; ChIP, chromatin immunoprecipitation; VHH, variable domain of heavy chain antibody; HEK, human embryonic kidney; IgG, immunoglobulin G; YFP, enhanced yellow fluorescent protein; CFP, enhanced cyan fluorescent protein; DsRed, Discosoma genus red fluorescent protein; mRFP, monomeric red fluorescent protein; PCNA, proliferating cell nuclear antigen; H2B, histone H2B; Dnmt1, DNA methyltransferase I; PBD, PCNA binding domain; HMGA1a, high mobility group protein A1a; Igf, insulin-like growth factor; PML, promyelocytic leukemia protein. and spectral variants thereof became popular tools to determine protein localization and, in combination with fluorescence photobleaching techniques, provided unique information on protein dynamics in living cells (1Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5451) Google Scholar, 2Misteli T. Spector D.L. Applications of the green fluorescent protein in cell biology and biotechnology.Nat. Biotechnol. 1997; 15: 961-964Crossref PubMed Scopus (307) Google Scholar, 3Tsien R.Y. The green fluorescent protein.Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4882) Google Scholar, 4van Roessel P. Brand A.H. Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins.Nat. Cell Biol. 2002; 4: E15-E20Crossref PubMed Scopus (211) Google Scholar). Necessary additional information on DNA binding, enzymatic activity, and complex formation can be obtained with various methods including chromatin immunoprecipitation (ChIP) and affinity and of 2002; PubMed Scopus Google Scholar, of chromatin in vivo 1997; PubMed Scopus Google Scholar). are the of specific are the protein of to specific protein including for PubMed Scopus Google Scholar, of protein from and biochemical to Biotechnol. PubMed Scopus Google Scholar). GFP, the most widely used in cell is used for biochemical various and of fluorescent protein to study nuclear of the in living Biol. 15: PubMed Scopus Google Scholar, proteins as 4: PubMed Scopus Google Scholar). This be in to and as as heavy and with to are variable single domain also to as VHH, derived from heavy chain of T. G. of PubMed Scopus Google Scholar). the with a mass of are and and can be produced in the Biol. PubMed Scopus Google Scholar, domain Biotechnol. Google Scholar). used for various P. in the of the human PubMed Scopus Google Scholar, of enzyme function in Biotechnol. PubMed Scopus Google Scholar, P. P. of specific for the domain in PubMed Scopus Google Scholar). we a of a 13-kDa GFP binding fragment derived from a llama single chain and in cells with fluorescent PubMed Scopus Google Scholar). This GFP-binding protein (GBP) a and can easily be produced in We immobilized the GBP to a enables a fast and efficient isolation of GFP fusion proteins and their interacting factors for biochemical and Moreover we the GFP-binding protein can be fused with proteins to ectopically recruit GFP fusion proteins and interacting factors in living cells. the localization and binding dynamics of fluorescent fusion proteins be with biochemical on interacting enzymatic activity, and DNA binding a for fast and efficient of GFP fusion proteins we used a 13-kDa GBP the domain of a heavy chain in GFP and in cells with fluorescent PubMed Scopus Google Scholar). The GBP fused with a produced in and a single the GBP can easily be produced and as a a functional we the binding of the GBP to of proteins and for the GBP and into a complex with a mass of We the of GBP to from cell and with and GBP and to protein of cells expressing proteins and After with GBP protein and the GBP additional to the and heavy of the as as proteins be in immunoprecipitations with the and The of GFP in to the GBP is efficient and and GBP can be immobilized and with protein to of the GBP and to with analyses we coupled GBP to a GFP-binding This GFP-nanotrap a fast of GFP from cell a single protein protein Moreover the a of the as of GFP from the the of GBP we immunoprecipitation with a of fluorescent proteins. GFP GBP the yellow of mRFP, further the binding of GBP we and we the of the binding from to and GBP high we and of GFP the and binding of we the of GFP in the range from to the to of GFP a fast and efficient to from the GFP-nanotrap GBP also GFP fusion proteins we from M. M. G. G. dynamics cell and a fusion Biol. 1997; PubMed Scopus Google G. and a in Cell Biol. PubMed Scopus Google P. T. of DNA in living Cell Biol. PubMed Scopus Google and T. fusion protein enables of dynamics in living Biol. 1998; PubMed Scopus Google Scholar). expressing the fusion proteins to determine the localization of the GFP fusion protein cells and GFP fusion proteins with the GFP-nanotrap and lower The GBP GFP fusion proteins from and of the most of the GBP is the to localization, and complex formation of specific proteins and We a of the PCNA domain chromatin of and PubMed Scopus Google Scholar). with the fluorescent with the red fluorescent PCNA We from living cells with biochemical and to immunoprecipitation with the GFP-nanotrap. with with lower The the of the in of DNA methyltransferase activity in living PubMed Scopus Google Scholar). we also PCNA with with GBP can also interacting proteins. the GFP-nanotrap is suitable to interacting factors and cell of and specific binding we the GFP-nanotrap to interacting factors mass in cell is the fluorescent fusion proteins are and biochemical to their fast of enzymatic activity we fluorescent fusion of the Dnmt1, are in cell we the and After of the fluorescent in of cells proteins from cell with the GFP-nanotrap and for enzymatic measurements. a specific DNA methyltransferase activity to of lower The with the the DNA methyltransferase of cells the GFP-nanotrap is a efficient for and biochemical of fluorescent fusion fusion proteins their enzymatic activity with the GFP-nanotrap. of from cells the Dnmt1, protein one with the GFP-nanotrap and DNA methyltransferase activity of GFP fusion proteins and the GFP-nanotrap the to function in chromatin immunoprecipitations we a cell expressing a GFP fusion of the high mobility group protein and We the of the and protein are to M. components chromatin Biol. PubMed Google Scholar, nuclear and high mobility group proteins the of the insulin-like growth 1997; PubMed Scopus Google Scholar, G. M. of gene function embryonic cell PubMed Scopus Google Scholar, M. G. of the and in and PubMed Scopus Google Scholar). of to immunoprecipitation with the GFP-nanotrap and with the GFP-nanotrap a of This binding of the specific with The cells expressing specific of in with the the high and of the with the GFP-nanotrap. of cells to immunoprecipitation of of and of DNA of the and and obtained for the and are in the obtained in as the obtained with the GFP-nanotrap and in vitro of the GFP-binding protein. a we in vivo and the of the GFP-binding protein a the cell to a of the nuclear We fused the GBP with to a cellular the nuclear GBP and the in vitro biochemical with binding in living cells we cells with GFP, and and the of GFP and with to the GFP-nanotrap the nuclear GFP the nuclear a the cell of GFP, YFP, and from the fluorescent proteins the GBP we single to We the of to binding to the to a binding This the high and of the GBP in vitro and in vivo allowing of single we the GFP-nanotrap can be used to factors and cellular a we the The is in nuclear a in regulation of gene DNA and P. and of Biol. PubMed Scopus Google Scholar, the promyelocytic leukemia protein for nuclear Biol. 2002; PubMed Scopus Google Scholar). We of in cells are the cells are the green fluorescence of the is for protein and cell is for the formation of Google fusion protein and with a specific expression of green fluorescent the nuclear and the of from the nuclear the of the GFP-nanotrap to recruit GFP fusion proteins also interacting of the in cells expressing GFP, CFP, and GFP is the nuclear is of GFP, CFP, and are in The of GFP The of green fluorescent Biotechnol. PubMed Scopus Google is and the is as a protein of cells expressing GFP, CFP, and to with the GFP-nanotrap. and and and with manipulation of nuclear structures with the GFP-nanotrap. cells in combination with are are on the is into with antibody. of the with to a of from the nuclear the of in the nuclear with the localization and binding dynamics of fluorescent fusion proteins be with biochemical on interacting enzymatic activity, and DNA binding a for fast and efficient of GFP fusion proteins we used a 13-kDa GBP the domain of a heavy chain in GFP and in cells with fluorescent PubMed Scopus Google Scholar). The GBP fused with a produced in and a single the GBP can easily be produced and as a a functional we the binding of the GBP to of proteins and for the GBP and into a complex with a mass of We the of GBP to from cell and with and GBP and to protein of cells expressing proteins and After with GBP protein and the GBP additional to the and heavy of the as as proteins be in immunoprecipitations with the and The of GFP in to the GBP is efficient and and GBP can be immobilized and with protein to of the GBP and to with analyses we coupled GBP to a GFP-binding This GFP-nanotrap a fast of GFP from cell a single protein protein Moreover the a of the as of GFP from the the of GBP we immunoprecipitation with a of fluorescent proteins. GFP GBP the yellow of mRFP, further the binding of GBP we and we the of the binding from to and GBP high we and of GFP the and binding of we the of GFP in the range from to the to of GFP a fast and efficient to from the GFP-nanotrap GBP also GFP fusion proteins we from M. M. G. G. dynamics cell and a fusion Biol. 1997; PubMed Scopus Google G. and a in Cell Biol. PubMed Scopus Google P. T. of DNA in living Cell Biol. PubMed Scopus Google and T. fusion protein enables of dynamics in living Biol. 1998; PubMed Scopus Google Scholar). expressing the fusion proteins to determine the localization of the GFP fusion protein cells and GFP fusion proteins with the GFP-nanotrap and lower The GBP GFP fusion proteins from and of the most of the GBP is the to localization, and complex formation of specific proteins and We a of the PCNA domain chromatin of and PubMed Scopus Google Scholar). with the fluorescent with the red fluorescent PCNA We from living cells with biochemical and to immunoprecipitation with the GFP-nanotrap. with with lower The the of the in of DNA methyltransferase activity in living PubMed Scopus Google Scholar). we also PCNA with with GBP can also interacting proteins. the GFP-nanotrap is suitable to interacting factors and cell of and specific binding we the GFP-nanotrap to interacting factors mass in cell is the fluorescent fusion proteins are and biochemical to their fast of enzymatic activity we fluorescent fusion of the Dnmt1, are in cell we the and After of the fluorescent in of cells proteins from cell with the GFP-nanotrap and for enzymatic measurements. a specific DNA methyltransferase activity to of lower The with the the DNA methyltransferase of cells the GFP-nanotrap is a efficient for and biochemical of fluorescent fusion proteins. the GFP-nanotrap the to function in chromatin immunoprecipitations we a cell expressing a GFP fusion of the high mobility group protein and We the of the and protein are to M. components chromatin Biol. PubMed Google Scholar, nuclear and high mobility group proteins the of the insulin-like growth 1997; PubMed Scopus Google Scholar, G. M. of gene function embryonic cell PubMed Scopus Google Scholar, M. 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The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.

Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.

Abstract Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC 50 -values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay.

Immunotherapy has become an established pillar of cancer treatment improving the prognosis of many patients with a broad variety of hematological and solid malignancies. The two main drivers behind this success are checkpoint inhibitors (CPIs) and chimeric antigen receptor (CAR) T cells. This review summarizes seminal findings from clinical and translational studies recently presented or published at important meetings or in top-tier journals, respectively. For checkpoint blockade, current studies focus on combinational approaches, perioperative use, new tumor entities, response prediction, toxicity management and use in special patient populations. Regarding cellular immunotherapy, recent studies confirmed safety and efficacy of CAR T cells in larger cohorts of patients with acute lymphoblastic leukemia or diffuse large B cell lymphoma. Different strategies to translate the striking success of CAR T cells in B cell malignancies to other hematological and solid cancer types are currently under clinical investigation. Regarding the regional distribution of registered clinical immunotherapy trials a shift from PD-1 / PD-L1 trials (mainly performed in the US and Europe) to CAR T cell trials (majority of trials performed in the US and China) can be noted.

Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy.

The potential of peptides as drug candidates is limited by their poor pharmacokinetic properties. Many peptides have a short half-life in vivo and a lack of oral availability. Inspired by the excellent pharmacokinetic profile of cyclosporine, a natural, multiply N-methylated cyclic peptide, we envisioned multiple N-methylation as a promising way to rationally improve key pharmacokinetic characteristics. In this Account, we summarize our efforts toward modulating the properties of peptides by multiple N-methylation. As a first step, we simplified the synthesis of N-methylated amino acids in solution, by employing very mild conditions that could be tolerated by the diverse protecting groups required when working with naturally occurring amino acids. We also report the rapid and inexpensive syntheses of N-methylated peptides on a solid support; this facilitated the N-methyl scanning of bioactive peptides. Because of a lack of information regarding the conformational behavior of multiply N-methylated peptides, a complete library of N-methylated cyclic alanine pentapeptides was synthesized. The library provided valuable insight into the conformational modulation of cyclic peptides by N-methylation. This information is extremely valuable for the design of bioactive peptides and spatial screening of cyclic N-methylated peptides. To demonstrate the applicability of N-methylation to highly active but poorly bioavailable peptides, we performed a full N-methyl scan of the cyclopeptidic somatostatin analog cyclo(-PFwKTF-), known as the Veber-Hirschmann peptide. We show here for the first time that the simple approach of multiple N-methylation can drastically improve the metabolic stability and intestinal permeability of peptides, for example, resulting in 10% oral bioavailability for a tri-N-methylated Veber-Hirschmann peptide analog. In addition, we also describe a designed approach to N-methylated peptide library synthesis, which can accelerate the screening of N-methylated bioactive peptides. Finally, we find that multiple N-methylation of a cyclic hexapeptide integrin antagonist of GPIIb-IIIa (alphaIIb beta3 integrin), cyclo(-GRGDfL-), increases the selectivity of this peptide toward different integrin subtypes. This result demonstrates the utility of multiple N-methylation in elucidating the bioactive conformation of peptides.

Hsp90 was originally identified as one of several conserved heat shock proteins. Like the other major classes of heat shock proteins, Hsp90 exhibits general protective chaperone properties, such as preventing the unspecific aggregation of non-native proteins (1Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (417) Google Scholar). However, Hsp90 seems to be more selective than the other promiscuous general chaperones, as it preferentially interacts with a specific subset of the proteome (2Picard D. CMLS Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar). Another specific feature of Hsp90 is its regulatory role of inducing conformational changes in folded, native-like substrate proteins that lead to their activation or stabilization (3Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Recently, the three-dimensional structures of full-length Hsp90 from Escherichia coli, yeast, and the endoplasmic reticulum were solved (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 7Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar). Together with sequence data, these showed that, although Hsp90 maintained its general domain structure from bacteria to man, distinct changes seem to have adapted Hsp90 to the more complex protein environment of the eukaryotic cell. Concomitant with the occurrence of a long charged linker connecting the N- 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. and M-domains, the eukaryotic protein exhibits an extension of the C-terminal domain, which includes the conserved amino acid motif MEEVD at the C terminus (8Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar). This region serves as the major interaction site for a cohort of co-chaperones (Table 1) (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar), which apparently support Hsp90 in the folding and activation of its substrate proteins in eukaryotes. In this review, we summarize the current knowledge on the functional principles of this molecular machine, including the ATP-driven chaperone cycle of Hsp90 and its regulation by co-chaperones and post-translational modifications.TABLE 1Selected Hsp90 cofactors Open table in a new tab Hsp90 is a flexible dimer. Each monomer consists of three domains: the N-domain, connected by a long linker sequence (in eukaryotes) to an M-domain, which is followed by a C-terminal dimerization domain (Fig. 1). The N-domain possesses a deep ATP-binding pocket (10Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), where ATP is bound in an unusual kinked manner. ATP hydrolysis by Hsp90 is rather slow: Hsp90 from yeast hydrolyzes one molecule of ATP every 1 or 2 min (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 12Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar), and human Hsp90 hydrolyzes one molecule of ATP every 20 min (0.04 min–1) (13McLaughlin S.H. Smith H.W. Jackson S.E. J. Mol. Biol. 2002; 315: 787-798Crossref PubMed Scopus (210) Google Scholar). The ATPase activity is essential for the function of Hsp90 in yeast (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 14Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (479) Google Scholar). The slow hydrolysis suggests that complex conformational rearrangements of Hsp90 are coupled to the ATPase reaction and that these represent the rate-limiting step of the enzyme. The first steps of these conformational changes were elucidated recently in detail (15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar): upon ATP binding, a short segment of the N-domain called the “ATP lid” changes its position and flaps over the binding pocket (Fig. 1, steps 2 and 3). This releases a short N-terminal segment from its original position (16Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In a subsequent reaction, this segment binds to the respective N-domain of the other subunit in the dimer, producing a strand-swapped, transiently dimerized N-terminal conformation (step 3) (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These N-terminal rearrangements result in further conformational changes throughout the entire Hsp90 dimer leading to a twisted and compacted dimer, in which N- and M-domains associate and the distance between M-domains is shortened by 40 Å (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar). The association of N- and M-domains completes the active site of this “split ATPase” (step 4). Recently, a similar progression of steps was shown to occur also for the endoplasmic homolog Grp94 (17Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), mitochondrial TRAP1 (6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 18Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J., and Reinstein, J. (2008) 283, 11677–11688Google Scholar), and human Hsp90 (19Richter K. Soroka J. Skalniak L. Leskovar A. Hessling M. Reinstein J. Buchner J. J. Biol. Chem. 2008; 283: 17757-17765Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Therefore, the scenario outlined above seems to be the ubiquitously conserved ATPase mechanism for Hsp90. Interestingly, the unusual way in which ATP is bound by Hsp90 is perfectly mimicked by some natural compounds, such as geldanamycin and radicicol. These are highly specific and potent inhibitors of the Hsp90 ATPase (20Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (871) Google Scholar), blocking the maturation of substrate proteins and eventually resulting in their degradation (21Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1310) Google Scholar). As several Hsp90 substrate proteins are kinases, which can be deregulated in the development of cancer, derivatives of Hsp90 inhibitors are currently being investigated as anticancer therapeutics at the stage of clinical trials (22Sharp S. Workman P. Adv. Cancer Res. 2006; 95: 323-348Crossref PubMed Scopus (269) Google Scholar). Current models assume that the conformational changes associated with ATP hydrolysis are required for reaching or maintaining an activated state of a substrate protein. In well studied examples such as the SHRs, several cofactors interact with Hsp90 in a sequential manner to assemble a functional chaperone machinery (23Pratt W.B. Toft D.O. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). The basis for this ordered succession of different assemblies can now be rationalized, as it turned out that several Hsp90 cofactors display a strong binding preference for specific Hsp90 conformations. The loading of an SHR onto Hsp90 requires the cooperation of Hsp90 with the chaperone Hsp70 and its cofactor Hsp40 (25Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar). Moreover, both chaperones become physically linked by an adaptor protein called Hop/Sti1 (Table 1). This co-chaperone binds via small helical TPR domains to the C-terminal ends of Hsp70 and Hsp90 (26Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). It seems that Hsp70 stabilizes the SHR in a conformation that can be recognized and bound by Hsp90. However, experimental evidence for this notion is largely lacking. How the substrate in this complex is transferred from Hsp70 to Hsp90 is also still unclear. It might be that the bridging by Hop/Sti1 selects for Hsp90 molecules in a conformation competent for substrate binding in addition to increasing the local concentration of Hsp70 and Hsp90. For the progression of the chaperone cycle, empty Hsp70 and Hop/Sti1 have to dissociate, and other co-chaperones such as specific PPIases and p23/Sba1 enter the complex (Table 1) (24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). These PPIases also possess a TPR domain, which binds to the C-terminal end of Hsp90. The second cofactor, p23/Sba1, associates with the N-terminally dimerized conformation of Hsp90 (27Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar), making it likely that the dramatic conformational rearrangement from the open to the closed state of Hsp90 occurs at this stage of the Hsp90 chaperone cycle (Fig. 1, steps 3 and 4). This closed conformation is metastable and upon ATP hydrolysis returns to the open state (Fig. 1, step 5) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The bound substrate protein dissociates in turn from Hsp90, permitting a new round of the cycle. The first steps of the cycle for the maturation of signaling kinases is a variation of the scheme described above. Here, the kinase-specific Hsp90 cofactor Cdc37 seems to associate with substrate kinases in their inactive forms first. This complex may then be loaded onto Hsp90 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. S. 1997; PubMed Scopus Google Scholar). The steps are It may be that also for kinases, and Hop/Sti1 are required A.K. Cell Biol. 2007; 17: Full Text Full Text PDF PubMed Scopus Google Scholar). Cdc37 the ATPase of Hsp90 G. Panaretou B. Meyer P. S. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), it is to that a of the ATPase substrate onto Hsp90 in to more than a distinct Hsp90 cofactors have identified (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar). is by other chaperone Hsp90 with (Table 1). The major of these is the TPR which the proteins and the (Table 1). of these cofactors the activation of a of substrate proteins. In this the cofactor shown to in development the of Brinker A. Hartl F.U. 2002; PubMed Scopus Google Scholar), and in complex with the protein from the N. C. W.M. Res. PubMed Scopus Google or the 1999; PubMed Scopus Google Scholar). It to be these cofactors are highly or are the first Interestingly, in yeast, cofactors of the Hsp90 are essential for (in addition to These are Cdc37 and (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. S. 1997; PubMed Scopus Google Scholar, J. Mol. Cell. Biol. 1998; PubMed Google Scholar, Mol. Cell. Biol. 1998; PubMed Google Scholar). shown to associate with both Hsp90 and to the of a TPR domain, can be O. H. K. Buchner J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The function of Hsp90 is several that its As the ATPase activity is to slow conformational changes of the segment the In addition to the ATPase activity of Hsp90 is by several Another of cofactors substrate the ATPase the activity of Hsp90 is also by post-translational of the cofactors of Hsp90 its ATPase activity by preferentially with a specific conformation of Hsp90. p23/Sba1 binds to the ATPase domain and stabilizes the N-terminally dimerized conformation at the stage of the ATPase cycle (Fig. 1) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar, A. I. Sullivan B. N. Toft D.O. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, G. B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This p23/Sba1 to be of the Hsp90 complex at the of hydrolysis and to be the for the in the ATP of Hsp90 in the of second site of cofactor interaction in the M-domain, to which the of the Hsp90 This interaction the ATPase activity of Hsp90 by B. Siligardi G. Meyer P. A. Sullivan S. S.H. S. R. R. M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction of with the of Hsp90 suggests a of this domain in the rate-limiting step of the ATPase that binding the the active and the domain to a conformation the closed for ATP hydrolysis 1, step 3) L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar, P. Prodromou C. C. B. Roe S.M. Vaughan C.K. I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; PubMed Scopus Google Scholar). Another for the ATPase activity of Hsp90 is by the co-chaperone in binds to the C-terminal end of Hsp90 via its TPR In is a second binding site in the N- or K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). to this second site to the ATPase of Hsp90 K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Siligardi G. O'Brien R. L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; PubMed Scopus Google Scholar). Interestingly, ATP binding is K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). As in the of p23/Sba1, selects a specific conformation of Hsp90 for suggests that binding the N-terminal dimerization and association of the N- and (Fig. 1, step 3) K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of ATP hydrolysis is is This is the of a The open state by is the state for as outlined above. Cdc37 also the ATPase activity of Hsp90 G. Panaretou B. Meyer P. S. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, C. Siligardi G. O'Brien R. L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; PubMed Scopus Google Scholar). of the respective are still a in which Cdc37 binds to the ATP in the N-terminal site of Hsp90 and the N-terminal dimerization by as a dimer in between the S.M. M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). 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We herein present a novel platform of well-controlled ordered and disordered nanopatterns positioned with a cyclic peptide of arginine-glycine-aspartic acid (RGD) on a bioinert poly(ethylene glycol) background, to study whether the nanoscopic order of spatial patterning of the integrin-specific ligands influences osteoblast adhesion. This is the first time that the nanoscale order of RGD ligand patterns was varied quantitatively, and tested for its impact on the adhesion of tissue cells. Our findings reveal that integrin clustering and such adhesion induced by RGD ligands is dependent on the local order of ligand arrangement on a substrate when the global average ligand spacing is larger than 70 nm; i.e., cell adhesion is "turned off" by RGD nanopattern order and "turned on" by the RGD nanopattern disorder if operating at this range of interligand spacing.