Moritz Marcinowski

Publications of Moritz Marcinowski

• Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions.

  Authors: Moritz Marcinowski, Matthias Höller, Matthias J Feige, Danae Baerend, Don C Lamb, Johannes Buchner

  Nature structural & molecular biology. 02/2011; 18(2):150-8.

  The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), togetherThe endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered C(H)1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Nature. 01/2011; 469:419-423.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of −200 mV to –300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Keystone Symposium, Mucosal Biology: A Fine Balance between Tolerance and Immunity, Vancouver, British Columbia; 01/2011

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1.

  Authors: Bjoern O Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F Stange, Jan Wehkamp

  Nature. 01/2011; 469(7330):419-23.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• An unfolded CH1 domain controls the assembly and secretion of IgG antibodies.

  Authors: Matthias J Feige, Sandra Groscurth, Moritz Marcinowski, Yuichiro Shimizu, Horst Kessler, Linda M Hendershot, Johannes Buchner

  Molecular cell. 07/2009; 34(5):569-79.

  A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are activelyA prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the C(H)1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain C(L) domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the C(H)1 domain and competes for binding to the C(L) domain. In vivo experiments demonstrate that requirements identified for folding the C(H)1 domain in vitro, including association with a folded C(L) domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.

• The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity.

  Authors: Matthias J Feige, Sandra Groscurth, Moritz Marcinowski, Zu Thur Yew, Vincent Truffault, Emanuele Paci, Horst Kessler, Johannes Buchner

  Proceedings of the National Academy of Sciences of the United States of America. 10/2008; 105(36):13373-8.

  Folding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessibleFolding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate exhibits the basic beta-barrel topology, yet some strands are distorted. Surprisingly, two short strand-connecting helices conserved in constant antibody domains assume their completely native structure already in the intermediate, thus providing a scaffold for adjacent strands. By transplanting these helical elements into beta(2)-microglobulin, a highly homologous member of the same superfamily, we drastically reduced its amyloidogenicity. Thus, minor structural differences in an intermediate can shape the folding landscape decisively to favor either folding or misfolding.

• Processing of proteins by the molecular chaperone Hsp104.

  Authors: Andreas Schaupp, Moritz Marcinowski, Valerie Grimminger, Benjamin Bösl, Stefan Walter

  Journal of molecular biology. 08/2007; 370(4):674-86.

  The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotideThe molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.

• Processing of Proteins by the Molecular Chaperone Hsp104

  Authors: Andreas Schaupp, Moritz Marcinowski, Valerie Grimminger, Benjamin Bösl, Stefan Walter

  Journal of Molecular Biology.

  The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotideThe molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.