FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds
Mühlberg(*), M., Hoesl(*), M. G., Kuehne(*), C., Dernedde(*), J., Budisa(*), N.; Hackenberger, C. P.
Beilstein J Org Chem, 11:784-791

Tags: Chemical Biology II (Hackenberger)

Abstract: To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and beta-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability
Nischan, N., Herce(*), H. D., Natale(*), F., Bohlke(*), N., Budisa(*), N., Cardoso(*), M. C.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 54:1950-1953

Tags: Chemical Biology II (Hackenberger)

Abstract: The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation
Nillegoda(*), N. B., Kirstein, J., Szlachcic(*), A., Berynskyy(*), M., Stank(*), A., Stengel(*), F., Arnsburg, K., Gao(*), X., Scior, A., Aebersold(*), R., Guilbride(*), D. L., Wade(*), R. C., Morimoto(*), R. I., Mayer(*), M. P.; Bukau(*), B.
Nature, 524:247-251

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation
Reuter(*), C., Opitz, R., Soicke(*), A., Dohmen(*), S., Barone, M., Chiha(*), S., Klein(*), M. T., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Chemistry, 21:8464-8470

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs
Planells-Cases, R., Lutter, D., Guyader(*), C., Gerhards(*), N. M., Ullrich, F., Elger, D. A., Kucukosmanoglu(*), A., Xu(*), G., Voss, F. K., Reincke, S. M., Stauber, T., Blomen(*), V. A., Vis(*), D. J., Wessels(*), L. F., Brummelkamp(*), T. R., Borst(*), P., Rottenberg(*), S.; Jentsch, T. J.
EMBO J, 34:2993-3008

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5
Protze, J., Eichner, M., Piontek, A., Dinter, S., Rossa, J., Blecharz(*), K. G., Vajkoczy(*), P., Piontek(*), J.; Krause, G.
Cellular and molecular life sciences : CMLS, 72:1417-1432

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 +/- 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Applying Acylated Fucose Analogues to Metabolic Glycoengineering
Rosenlöcher(*), J., Böhrsch, V., Sacharjat(*), M., Blanchard(*), V., Giese(*), C., Sandig(*), V., Hackenberger, C.; Hinderlich(*), S.
Bioengineering, 2:213

Tags: Chemical Biology II (Hackenberger)

Optogenetic acidification of synaptic vesicles and lysosomes
Rost(*), B. R., Schneider(*), F., Grauel(*), M. K., Wozny(*), C., Bentz(*), C. G., Blessing, A., Rosenmund(*), T., Jentsch, T. J., Schmitz(*), D., Hegemann(*), P.; Rosenmund(*), C.
Nat Neurosci, 18:1845-1852

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate
Schotten(*), S., Meijer(*), M., Walter, A. M., Huson(*), V., Mamer(*), L., Kalogreades(*), L., ter Veer(*), M., Ruiter(*), M., Brose(*), N., Rosenmund(*), C., Sorensen(*), J. B., Verhage(*), M.; Cornelisse(*), L. N.
Elife, 4:e05531

Tags: Molecular and Theoretical Neuroscience (Walter)

Abstract: The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca(2+)-dependent release.

Cohen syndrome-associated protein COH1 physically and functionally interacts with the small GTPase RAB6 at the Golgi complex and directs neurite outgrowth
Seifert(*), W., Kühnisch(*), J., Maritzen, T., Lommatzsch, S., Hennies, H. C., Bachmann, S., Horn, D.; Haucke, V.
J Biol Chem, 290:3349-3358

Tags: Molecular Pharmacology and Cell Biology (Haucke), Membrane Traffic and Cell Motility (Maritzen)

Abstract: Postnatal microcephaly, intellectual disability, and progressive retinal dystrophy are major features of autosomal recessive Cohen syndrome, which is caused by mutations in the gene COH1 (VPS13B). We have recently identified COH1 as a Golgi-enriched scaffold protein that contributes to the structural maintenance and function of the Golgi complex. Here, we show that association of COH1 with the Golgi complex depends on the small GTPase RAB6. RNAi-mediated knockdown of RAB6A/A' prevents the localization of COH1 to the Golgi complex. Expression of the constitutively inactive RAB6_T27N mutant led to an increased solubilization of COH1 from lipid membrane preparations. Co-IP experiments confirmed the physical interaction of COH1 with RAB6 that preferentially occurred with the constitutively active RAB6_Q72L mutants. Depletion of COH1 in primary neurons negatively interfered with neurite outgrowth, indicating a causal link between the integrity of the Golgi complex and axonal outgrowth. We conclude that COH1 is a RAB6 effector protein and that reduced brain size in Cohen syndrome patients likely results from impaired COH1 function at the Golgi complex, causing decreased neuritogenesis.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
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