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In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.
Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+), cytotoxic-T cells (CD8+), T-helper cells (CD4+), B cells (CD20+), macrophages (CD68+), mast cells (CD117+), mononuclear cells (CD11c+), plasma cells, activated-T cells (MUM1+), B cells, myeloid cells (PD1+) and neutrophilic granulocytes (myeloperoxidase+) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.
Hepatitis B caused by infection with the hepatitis B virus (HBV) still ranks among the most challenging infectious diseases of our time. Despite the availability of an effective prophylactic vaccine, 240 million people worldwide are estimated to be chronically infected with HBV and are at risk of developing life-threatening liver diseases, including cirrhosis and liver cancer. The underlying pathogenic mechanisms of HBV-associated liver diseases are only incompletely understood. It is widely accepted that liver pathology results from long-term immune-mediated liver injury and inflammation as a consequence of inefficient viral elimination. This injury can be naturally compensated by liver regeneration. However, chronic liver damage and permanent inflammation debilitates the regenerative capacity of the liver and fosters fibrosis as well as accumulation of chromosomal aberrations, which both contribute to cirrhosis and liver cancer. Liver regeneration requires the presence of the redox-sensitive transcription factor Nrf2 and intact insulin receptor signaling. A lack of Nrf2 causes increased intracellular levels of reactive oxygen species (ROS) that inactivate insulin receptor signaling and induce insulin resistance. Interestingly, HBV was observed to activate Nrf2 and the expression of Nrf2-regulated genes. This argues against an inhibitory effect of HBV on insulin receptor signaling by increased ROS levels. However, chronic HBV infection is associated with dysregulation of hepatocyte proliferation and retardation of liver regeneration. Hence, the aim of this thesis was to investigate the influence of HBV on the process of liver regeneration with respect to the insulin receptor signaling pathway. After short-term carbon tetrachloride (CCl4)-induced liver damage, HBV transgenic mice present prolonged liver damage and impaired liver regeneration as reflected by reduced hepatocyte proliferation and increased apoptosis. Impaired hepatocyte proliferation in HBV transgenic mice correlates with diminished activation of the insulin receptor. It was further observed in vitro that the activation of Nrf2 by HBV induces increased levels of the insulin receptor mRNA and protein in HBV-expressing cells. Strikingly, stably HBV-expressing cells as well as primary mouse hepatocytes from HBV transgenic mice bind less insulin due to reduced amounts of insulin receptor on the cell surface. This is caused by intracellular retention of the insulin receptor in HBV-expressing cells as a consequence of increased amounts of the cellular trafficking factor α-taxilin. The reduced amounts of insulin receptor on the cell surface impair insulin sensitivity in HBV-expressing cells and inactivate downstream signaling cascades that initiate insulin-dependent gene expression and glucose uptake. As a consequence of impaired hepatocyte proliferation and liver regeneration, HBV transgenic mice exhibit increased development of fibrosis after long-term CCl4-induced liver damage. Taken together, in this thesis, a novel pathomechanism could be uncovered that includes inactivation of insulin receptor signaling by HBV via intracellular retention of the insulin receptor leading to impaired liver regeneration after liver damage and promotion of liver fibrosis. These findings significantly contribute to an enhanced understanding of HBV-associated liver pathogenesis.
The knowledge of three-dimensional structures of biomolecules is fundamental for the understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy represents besides X-ray crystallography one of the two most widely used techniques to study macromolecules at atomic resolution. Its application has long been a laborious task that could take months and required the expertise of an experienced scientist, however, owing to the tremendous effort that has been put into the development of respective computer algorithms, structure determination by NMR spectroscopy of small- to medium sized proteins is nowadays routinely performed. CYANA is one widely used software package, which combines the majority of individual steps towards a three-dimensional structure. The most common application of the program, however, restricts to the combined automated NOE assignment and structure calculation based on NOESY peak lists and an existing chemical shift assignment. Completely automated structure determination starting from NMR spectra is to date technically possible with CYANA, however, not yet routinely applied. In order to achieve this long-term goal, the individual steps need to become more robust with regard to data imperfections such as peak overlap, spectral artifacts or a limited amount of NMR data. The work presented in this thesis should be placed within the context of increasing the reliability and improving the accuracy of structures determined by CYANA on the basis of solution- as well as solid-state NMR data.
The chapter “Systematic evaluation of combined automated NOE assignment and structure calculation with CYANA” comprises an extensive study on the robustness of the combined automated NOE assignment and structure calculation algorithm based on experimental solution NMR data sets that were modified in multiple ways to mimic different kinds of data imperfections. The results show that the algorithm is remarkably robust with regard to imperfections of the NOESY peak lists and the chemical shift tolerances but susceptible to lacking or erroneous resonance assignments, in particular for nuclei that are involved in many NOESY cross peaks.
In the chapter “Peakmatch – A simple and robust tool for peaklist matching” a method to achieve self-consistency of the chemical shift referencing among a set of peak lists is presented. The Peakmatch algorithm matches a set of peak lists to a specified reference peak list, neither of which have to be assigned, by optimizing an assignment-free match-score function. The algorithm has been extensively tested on the basis of experimental NMR data sets of five different proteins. The results show that peak lists from many different types of spectra can be matched reliably as long as they contain at least two corresponding dimensions.
NMR structures are represented by bundles of conformers whose spread indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e. how close NMR conformers are to the “true” structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase the precision thus often yield tight structure bundles where the precision overestimates the accuracy. To overcome this problem, the chapter “Increased reliability of NMR protein structures by consensus structure bundles” introduces a new protocol for NMR structure determination with the software package CYANA that produces bundles of conformers with a realistic precision that is throughout a large number of test data sets a much better estimate of the structural accuracy than the precision of conventional structure bundles.
Solid-state NMR is a powerful technique to study molecules which are not amenable to either solution NMR or X-ray crystallography. Despite the reporting of individual atomic resolution structures of membrane proteins and amyloid fibrils based on solid-state NMR data, the application is far from routine. One major obstacle that hinders structure determination by solid-state NMR is the overall lower quality of the solid-state NMR spectra. It is therefore necessary to increase the robustness of the computer algorithms in order to improve the results when using lower quality solid-state NMR spectra. The chapter “Structure calculations of the model protein GB1 from solid-state NMR data” presents structure calculations on the basis of a set of two-dimensional solid-state NMR experiments of the model protein GB1. The most important result obtained from these test calculations is that the limitation of structural accuracy can be attributed to inaccurate distance information resulting from the limited correlation between peak intensities and distance, which is especially severe in spin diffusion-based solid-state NMR experiments.
The chapter “Full relaxation matrix-based correction of relayed polarization transfer for solid-state NMR structure calculation” therefore introduces a method which corrects experimental peak intensities for spin diffusion in order to improve the distance information from solid-state NMR spectra. The results show that the structural accuracy can be significantly improved when using the corrected distance information, however, strongly dependent on the preliminary structural model that is required as input for the method.
Cardiac progenitor cells hold great potential for regenerative therapies in heart disorders. However, the molecular mechanisms regulating cardiac progenitor cell expansion and differentiation remain poorly defined. Here we show that the multi- adaptor protein Ldb1, which mediates interactions between different classes of LIM domain transcription factors, is a multifunctional regulator of cardiac progenitor cell differentiation. Ldb1-deficient embryonic stem cells (ESCs) show a markedly decreased expression of second heart field (SHF) marker genes and subsequently impaired cardiomyocyte differentiation. Conditional ablation of Ldb1 in the early SHF using an Isl1-Cre driver led to embryonic lethality at Embryonic day (E)10.5 with cardiac abnormalities including a significantly smaller right ventricle and a shortened outflow tract, supporting a crucial role of Ldb1 in the SHF. Mechanistically we show that the importance of Ldb1 for SHF development is two-fold: On the one hand, Ldb1 binds to Isl1 and protects it from proteasomal degradation, as a consequence of which Ldb1-deficiency leads to an almost complete loss of Isl1+ cardiovascular progenitor cells. On the other hand the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing (3C- seq) identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. These interactions are of critical importance to regulate the expression of the downstream target genes since their expression levels are strongly dependent on the Ldb1/Isl1 levels. Overexpression of an Ldb1 mutant, which contains the LIM interaction domain and thereby can protect Isl1 protein from degradation, but lacks the dimerization domain and thus cannot promote long-range interactions, does not collaborate with Isl1 to regulate the expression of their common targets and results in defects in Isl1+ cardiac progenitor differentiation. In this thesis we show one of the first examples of genome-wide chromatin reorganization mediated by a developmental regulated, cell type specific, transcription complex. Ldb1 in concert with Isl1 promotes long range promoter- enhancer and enhancer-enhancer interactions in order to create active chromatin hub where gene important for heart development can be co-regulated. Moreover, Isl1 and Ldb1 genetically interact during heart development, as Isl1/Ldb1 haplodeficient embryos show various cardiac anomalies. The dosage-sensitive interdependence between Isl1 and Ldb1 in the expression of these key factors in cardiogenesis, further supports a key role of the Isl1/Ldb1 complex in coordinating a three dimensional genome organization, upstream of a regulatory network driving cardiac differentiation and heart development.
In conclusion, the Isl1/Ldb1 complex orchestrate a genome-wide three dimensional chromatin reorganization resulting in a transcriptional program responsible for the differentiation of multipotent cardiac progenitor cells into cardiomyocytes.
Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice
(2015)
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I
(2015)
While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.
As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.