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Princz, L.N., Wild, P., Bittmann, J., Aguado, F.J., Blanco, M.G., Matos, J., and Pfander, B.
EMBO J, 2017, [Epub ahead of print].

Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.


 

Circadian is the latin meaning for “about a day”. Circadian clocks have evolved to adapt our lives to the daily environmental changes on earth: light and warmth during the day and darkness and cold at night. Scientists at the Max-Planck-Institute of Biochemistry in Martinsried discovered with the help of the mass spectrometry, that more than 25 percent of the molecular protein switches in mouse liver cells change in a daily manner. These rhythmic switches are binding sites for phosphate molecules, that regulate the function of proteins, and thereby the daily metabolic processes in the organ. The study was published in the journal Cell Metabolism.

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Bantele, S.C., Ferreira, P., Gritenaite, D., Boos, D., and Pfander, B.
Elife, 2017, 6.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection.

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold proteins Dpb11 and TOPBP1, respectively. In yeast this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice.


 

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Thanisch, K., Song, C., Engelkamp, D., Koch, J., Wang, A., Hallberg, E., Foisner, R., Leonhardt, H., Stewart, C.L., Joffe, B., and Solovei, I.
Differentiation, 2017, 94, 58-70.

Nuclear envelope localization of LEMD2 is developmentally dynamic and lamin A/C dependent yet insufficient for heterochromatin tethering.

Peripheral heterochromatin in mammalian nuclei is tethered to the nuclear envelope by at least two mechanisms here referred to as the A- and B-tethers. The A-tether includes lamins A/C and additional unknown components presumably INM protein(s) interacting with both lamins A/C and chromatin. The B-tether includes the inner nuclear membrane (INM) protein Lamin B-receptor, which binds B-type lamins and chromatin. Generally, at least one of the tethers is always present in the nuclear envelope of mammalian cells. Deletion of both causes the loss of peripheral heterochromatin and consequently inversion of the entire nuclear architecture, with this occurring naturally in rod photoreceptors of nocturnal mammals. The tethers are differentially utilized during development, regulate gene expression in opposite manners, and play an important role during cell differentiation. Here we aimed to identify the unknown chromatin binding component(s) of the A-tether. We analyzed 10 mouse tissues by immunostaining with antibodies against 7 INM proteins and found that every cell type has specific, although differentially and developmentally regulated, sets of these proteins. In particular, we found that INM protein LEMD2 is concomitantly expressed with A-type lamins in various cell types but is lacking in inverted nuclei of rod cells. Truncation or deletion of Lmna resulted in the downregulation and mislocalization of LEMD2, suggesting that the two proteins interact and pointing at LEMD2 as a potential chromatin binding mediator of the A-tether. Using nuclei of mouse rods as an experimental model lacking peripheral heterochromatin, we expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that in contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages.


 

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Hamzeiy, H., and Cox, J.
Curr Opin Biotechnol, 2016, 43, 141-146.

What computational non-targeted mass spectrometry-based metabolomics can gain from shotgun proteomics.

Computational workflows for mass spectrometry-based shotgun proteomics and untargeted metabolomics share many steps. Despite the similarities, untargeted metabolomics is lagging behind in terms of reliable fully automated quantitative data analysis. We argue that metabolomics will strongly benefit from the adaptation of successful automated proteomics workflows to metabolomics. MaxQuant is a popular platform for proteomics data analysis and is widely considered to be superior in achieving high precursor mass accuracies through advanced nonlinear recalibration, usually leading to five to ten-fold better accuracy in complex LC-MS/MS runs. This translates to a sharp decrease in the number of peptide candidates per measured feature, thereby strongly improving the coverage of identified peptides. We argue that similar strategies can be applied to untargeted metabolomics, leading to equivalent improvements in metabolite identification.


 

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Geyer, P.E., Wewer Albrechtsen, N.J., Tyanova, S., Grassl, N., Iepsen, E.W., Lundgren, J., Madsbad, S., Holst, J.J., Torekov, S.S., and Mann, M.
Mol Syst Biol, 2016, 12, 901.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10-13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.


 

Although the terms “cryo-EM” and “SKI complex” evoke images of ice and snow, they actually relate to structural biology. Scientists at the Max Planck Institute of Biochemistry and the Gene Center of the University of Munich (LMU) have now shown that the cellular protein factory and the SKI protein complex are in direct contact. The SKI complex is part of a molecular shredder that breaks down mRNA, the construction manual for proteins, into its individual components. To conduct their analysis, the researchers used cryo-electron microscopy, a technique that involves flash-freezing protein complexes to allow even the tiniest details of their structure to be studied in their natural state.

Ribosomes are the molecular protein factories of cells. Following a construction manual – the messenger RNA, or mRNA – they assemble protein building blocks to form chains. These chains are later folded into tiny molecular machines – the proteins. Proteins then perform a variety of tasks in the cells. Roland Beckmann and his team at the Gene Center of the University of Munich (LMU) specialize in investigating the structure of the ribosomes using cryo-electron microscopy. Elena Conti’s group “Structural Cell Biology” at the Max Planck Institute of Biochemistry in Martinsried has been studying the structure and function of the exosome, a shredder for mRNA molecules, for many years. When the protein construction manual is no longer needed, or when it contains an error, the exosome breaks down the mRNA into its basic components and recycles it.

In a joint project between these two groups, scientists at the two institutions have now shown that the SKI protein complex, which serves as the motor for the exosome in breaking down mRNA, is in direct contact with the ribosomes.

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Lemke, S.B., and Schnorrer, F.
Mech Dev, 2016,  [Epub ahead of print].

Mechanical forces during muscle development.


Muscles are the major force producing tissue in the human body. While certain muscle types specialise in producing maximum forces, others are very enduring. An extreme example is the heart, which continuously beats for the entire life. Despite being specialised, all body muscles share similar contractile mini-machines called sarcomeres that are organised into regular higher order structures called myofibrils. The major sarcomeric components and their organisational principles are conserved throughout most of the animal kingdom. In this review, we discuss recent progress in the understanding of myofibril and sarcomere development largely obtained from in vivo models. We focus on the role of mechanical forces during muscle and myofibril development and propose a tension driven self-organisation mechanism for myofibril formation. We discuss recent technological advances that allow quantification of forces across tissues or molecules in vitro and in vivo. Although their application towards muscle development is still in its infancy, these technologies are likely to provide fundamental new insights into the mechanobiology of muscle and myofibril development in the near future.


 

The immune system can fight specifically against cancer by tumor-specific T cells although suitable altered target structures are currently mostly unknown. A team at the the Max Planck Institute of Biochemistry and Technical University of Munich (TUM) has developed a method that allows for the first time the reliable identification of suitable antigens directly from patients` tumor cells by mass spectrometry. These structures proved to be immunogenic. This procedure therefore opens up new possibilities for individualized targeted cancer treatments.

Through evolution, the immune system has developed sophisticated mechanisms for fighting illnesses associated with viruses and tumors. T cells play an important role in this setting. They can identify peptides, small protein sturctures, presented by the body`s own cells that may indicate a viral infection or a genetic alteration as for example a mutation – a characteristic of tumor cells. Peptides identified by immune cells are known as antigens. T cells that recognize antigens can trigger a reaction that destroys the targeted cells. In recent years research teams, including TUM researchers, have successfully utilized this characteristic for cancer treatments. Different approaches have emerged. Vaccinating a patient with an antigen can stimulate the body to enhance the production of specific T cells. Another possibility is to enrich T cells that are "trained" for certain antigens and transfer them to the patient.

A method, that identifies the antigen structures of patient specific tumors has now been developed by a team led by Angela M. Krackhardt, a professor of translational immunotherapy at the Third Medical Clinic at TUM's Klinikum rechts der Isar, and Professor Matthias Mann of the Department of Proteomics and Signal Transduction at the Max Planck Institute of Biochemistry. Krackhardt and Mann have described their approach in an article published in the Journal Nature Communications. Unlike former applied methods, it is not based on predictive models. Instead, a mass spectrometer is used to identify the peptides actually present on the tumor surface.

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Falkner, S., Grade, S., Dimou, L., Conzelmann, K.K., Bonhoeffer, T., Gotz, M., and Hubener, M.
Nature, 2016, 539, 248-253.

Transplanted embryonic neurons integrate into adult neocortical circuits.

The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4-8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.