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Geyer PE, Holdt LM, Teupser D, Mann M.
Mol Syst Biol 2017, 13, 942.
doi: 10.15252/msb.20156297

Revisiting biomarker discovery by plasma proteomics.

Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and blood biomarkers are used to categorize patients and to support treatment decisions. However, existing biomarkers are far from comprehensive and often lack specificity and new ones are being developed at a very slow rate. As described in this review, mass spectrometry (MS)-based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous "triangular strategies" aimed at discovering single biomarker candidates in small cohorts, followed by classical immunoassays in much larger validation cohorts. We propose a "rectangular" plasma proteome profiling strategy, in which the proteome patterns of large cohorts are correlated with their phenotypes in health and disease. Translating such concepts into clinical practice will require restructuring several aspects of diagnostic decision-making, and we discuss some first steps in this direction.


 

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Pasalic D, Weber B, Giannone C, Anelli T, Müller R, Fagioli C, Felkl M, John C, Mossuto MF, Becker CFW, Sitia R, Buchner J.
Proc Natl Acad Sci USA 2017, [Epub ahead of print].
doi: 10.1073/pnas.1701797114

A peptide extension dictates IgM assembly.

Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-μ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension.


 

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Li L, Lingaraju M, Basquin C, Basquin J, Conti E
RNA 2017, 23, 1028-1034.
doi: 10.1261/rna.061200.117

Structure of a SMG8-SMG9 complex identifies a G-domain heterodimer in the NMD effector proteins.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA degradation pathway involved in surveillance and post-transcriptional regulation, and executed by the concerted action of several trans-acting factors. The SMG1 kinase is an essential NMD factor in metazoans and is associated with two recently identified and yet poorly characterized proteins, SMG8 and SMG9. We determined the 2.5 Å resolution crystal structure of a SMG8-SMG9 core complex from C. elegans We found that SMG8-SMG9 is a G-domain heterodimer with architectural similarities to the dynamin-like family of GTPases such as Atlastin and GBP1. The SMG8-SMG9 heterodimer forms in the absence of nucleotides, with interactions conserved from worms to humans. Nucleotide binding occurs at the G domain of SMG9 but not of SMG8. Fitting the GDP-bound SMG8-SMG9 structure in EM densities of the human SMG1-SMG8-SMG9 complex raises the possibility that the nucleotide site of SMG9 faces SMG1 and could impact the kinase conformation and/or regulation.


 

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Varga J & Greten FR
Nat Cell Biol, 2017, [Epub ahead of print].
doi: 10.1038/ncb3611

Cell plasticity in epithelial homeostasis and tumorigenesis

The adult organism is characterized by remarkable plasticity, which enables efficient regeneration and restoration of homeostasis after damage. When aberrantly activated, this plasticity contributes to tumour initiation and progression. Here we review recent advances in this field with a focus on cell fate changes and the epithelial-mesenchymal transition-two distinct, yet closely related, forms of plasticity with fundamental roles in homeostasis and cancer.


 

Proteins are often considered as molecular machines. To understand how they work, it is not enough to visualize the involved proteins under the microscope. Wherever machines are at work mechanical forces occur, which in turn influence biological processes. These extremely small intracellular forces can be measured with the help of molecular force sensors. Now researchers at the Max Planck Institute of Biochemistry in Martinsried have developed molecular probes that can measure forces across multiple proteins with high resolution in cells. The results of their work were published in the journal Nature Methods.

When proteins pull on each other, forces in the piconewton range are generated. Cells can detect such mechanical information and modulate their response depending on the nature of the signal. Adhesion proteins on the surface of cells, for instance, recognize how rigid their environment is to adjust the protein composition of the cell accordingly. To measure such tiny forces, the group of Molecular Mechanotransduction at the Max Planck Institute is developing molecular force sensors. “These small measuring instruments work along the lines of a spring scale,” says Carsten Grashoff, head of the research group.

The innovative probes consist of two fluorescent molecules that are connected by a sort of molecular spring. When a force of just a few piconewton acts on the molecule, the spring stretches, and this change can be detected using a special microscopic method. “We’re now able to measure the mechanics of several molecules simultaneously,” Carsten Grashoff explains. In contrast to previous experiments, the scientists are not only able to determine which proteins, but also how many of them are under force at any given moment.

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di Cicco G, Bantele SCS, Reusswig KU, Pfander B.
Sci Rep 7, 11650.
doi: 10.1038/s41598-017-11937-z

A cell cycle-independent mode of the Rad9-Dpb11 interaction is induced by DNA damage.

Budding yeast Rad9, like its orthologs, controls two aspects of the cellular response to DNA double strand breaks (DSBs) - signalling of the DNA damage checkpoint and DNA end resection. Rad9 binds to damaged chromatin via modified nucleosomes independently of the cell cycle phase. Additionally, Rad9 engages in a cell cycle-regulated interaction with Dpb11 and the 9-1-1 clamp, generating a second pathway that recruits Rad9 to DNA damage sites. Binding to Dpb11 depends on specific S/TP phosphorylation sites of Rad9, which are modified by cyclin-dependent kinase (CDK). Here, we show that these sites additionally become phosphorylated upon DNA damage. We define the requirements for DNA damage-induced S/TP phosphorylation of Rad9 and show that it is independent of the cell cycle or CDK activity but requires prior recruitment of Rad9 to damaged chromatin, indicating that it is catalysed by a chromatin-bound kinase. The checkpoint kinases Mec1 and Tel1 are required for Rad9 S/TP phosphorylation, but their influence is likely indirect and involves phosphorylation of Rad9 at S/TQ sites. Notably, DNA damage-induced S/TP phosphorylation triggers Dpb11 binding to Rad9, but the DNA damage-induced Rad9-Dpb11 interaction is dispensable for recruitment to DNA damage sites, indicating that the Rad9-Dpb11 interaction functions beyond Rad9 recruitment.


 

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Andreeva L, Hiller B, Kostrewa D, Lässig C, de Oliveira Mann CC, Jan Drexler D, Maiser A, Gaidt M, Leonhardt H, Hornung V, Hopfner KP.
Nature, 2017,  [Epub ahead of print].
doi: 10.1038/nature23890.

cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders.

Cytosolic DNA arising from intracellular pathogens triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP). Endogenous nuclear or mitochondrial DNA can also be sensed by cGAS under certain conditions, resulting in sterile inflammation. The cGAS dimer binds two DNA ligands shorter than 20 base pairs side-by-side, but 20-base-pair DNA fails to activate cGAS in vivo and is a poor activator in vitro. Here we show that cGAS is activated in a strongly DNA length-dependent manner both in vitro and in human cells. We also show that cGAS dimers form ladder-like networks with DNA, leading to cooperative sensing of DNA length: assembly of the pioneering cGAS dimer between two DNA molecules is ineffective; but, once formed, it prearranges the flanking DNA to promote binding of subsequent cGAS dimers. Remarkably, bacterial and mitochondrial nucleoid proteins HU and mitochondrial transcription factor A (TFAM), as well as high-mobility group box 1 protein (HMGB1), can strongly stimulate long DNA sensing by cGAS. U-turns and bends in DNA induced by these proteins pre-structure DNA to nucleate cGAS dimers. Our results suggest a nucleation-cooperativity-based mechanism for sensitive detection of mitochondrial DNA and pathogen genomes, and identify HMGB/TFAM proteins as DNA-structuring host factors. They provide an explanation for the peculiar cGAS dimer structure and suggest that cGAS preferentially binds incomplete nucleoid-like structures or bent DNA.


 

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Sahasrabudhe P, Rohrberg J, Biebl MM, Rutz DA, Buchner J.
Mol Cell, 2017, [Epub ahead of print].
doi: 10.1016/j.molcel.2017.08.004.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients. Only a few co-chaperones are active on all clients studied. Closely related clients and even point mutants can depend on different co-chaperones. These effects are direct because differences in client-co-chaperone interactions can be reconstituted in vitro. Interestingly, some co-chaperones affect client conformation in vivo. Thus, co-chaperones adapt the Hsp90 cycle to the requirements of the client proteins, ensuring optimal activation.


 

Multiple sclerosis (MS) is the most common inflammatory disease of the central nervous system. It has been suspected for some time that bacteria in the natural intestinal flora may be responsible for triggering the disease in individuals genetically predisposed to it. Together with researchers from the Ludwig Maximilian University of Munich, the Max Planck Institute of Immunobiology and Epigenetics in Freiburg and the Universities of California (San Francisco) and Münster, Hartmut Wekerle and Gurumoorthy Krishnamoorthy from the Max Planck Institutes of Neurobiology and of Biochemistry in Martinsried have, for the first time, shown that intestinal flora from patients with MS can trigger an MS-like illness in an animal model.

n autoimmune diseases such as multiple sclerosis, errant immune cells attack the body's own cells in the brain and spinal cord. Attacks triggered by autoimmune T-cells damage the nerve cells and result in the destruction of the sheath that surrounds these cells. The cells die off, with the result that nerve impulses are no longer transmitted correctly.

Every person has T-cells with the potential to attack their own cells; however, these cells usually remain permanently dormant. In some people, however, the pathogenic potential of these cells is activated, resulting in MS. Scientists believe that activation is caused by a combination of genetic and environmental factors. "More than 200 genes that increase susceptibility to MS have now been identified," explains Hartmut Wekerle, Hertie Professor and Emeritus Director at the Max Planck Institute of Neurobiology, “but for MS to develop, there must be a trigger. To date, most research on triggers has looked at infectious diseases.” A few years ago, the neuroimmunologist, together with his colleagues Kerstin Berer and Gurumoorthy Krishnamoorthy who now leads a Research Group at the MPI of Biochemistry, determined that this trigger is likely to be found in the natural intestinal flora. Together with other colleagues, the researchers showed that intestinal microorganisms were able to activate T-cells in genetically modified autoimmune mice, causing the mice to develop brain lesions similar to those found in MS.

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A common feature of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s is the accumulation of toxic protein deposits in the nerve cells of patients. Once these aggregates appear, they begin to proliferate like weeds. If and how these deposits damage nerve cells and lead to their demise remains largely unexplained. A detailed insight into the three-dimensional structure of the protein aggregates should help researchers to solve this puzzle. Now, using cryo-electron tomography, scientists at the Max Planck Institute of Biochemistry in Martinsried near Munich have succeeded in generating a high-resolution, three-dimensional model of the huntingtin aggregates responsible for Huntington’s disease. The results are published in the journal Cell.

Rampant weed growth – the nightmare of every hobby gardener. Trimming, cropping, cutting. Thorough garden maintenance is required. If this maintenance is neglected, weeds gain the upper hand and suppress the growth of crop and ornamental plants. The same applies to proteins in our bodies: molecular machines, large protein complexes that control vital cellular processes, assume the responsibility of a gardener. These molecular machines ensure that proteins reach their correct conformations and tend to and care for them for the duration of their lifespans.

A matter of the correct form
In order to carry out its function, a protein needs to adopt its correct three-dimensional structure. The building blocks of proteins, the amino acids, are assembled into long chains and folded into a complex form. If the resulting structure is faulty, the defective proteins are broken down in a strictly regulated process. If this does not occur properly, the misfolded proteins may aggregate forming clumps and deposits. Insoluble protein aggregates are toxic for cells. In the brain of patients suffering from neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or Huntington’s, protein aggregates are often found.

If and how exactly these aggregates exert their toxic effects has not yet been explained. This is the question studied by the ToPAG (Toxic Protein AGgregation in neurodegeneration) consortium. A team of researchers in the departments of Wolfgang Baumeister, Ulrich Hartl and Rüdiger Klein has succeeded in decoding a 3D structure of the protein aggregates linked to Huntington’s disease within their intact cellular environment.

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