Quantification of mitochondrial cfDNA reveals new perspectives for early diagnosis of colorectal cancer.

BACKGROUND: To unravel how the integrity of nuclear and mitochondrial circulating cell-free DNA (cfDNA) contributes to its plasma quantity in colorectal cancer (CRC) patients. METHODS: CfDNA from plasma samples of 80 CRC patients stratified by tumour stage and 50 healthy individuals were extracted. Total cfDNA concentration was determined and equal template concentrations (ETC) were analyzed by quantitative real-time PCR (qPCR) resulting in small and long fragments of KRAS, Alu and MTCO3. The obtained data was also examined relative to the total cfDNA concentration (NTC) and diagnostic accuracy was estimated using receiver operating characteristics. RESULTS: Total cfDNA levels were significantly higher in CRC group compared to healthy control and increased with tumour stage. Long nuclear fragment levels were significantly lower in CRC patients in ETC but not NTC condition. The integrity indices of nuclear cfDNA decreased from controls to patients with highly malignant tumor. Mitochondrial cfDNA fragment quantities were strongly reduced in early and late stages of tumor patients and prognostic value was higher in ETC. Predictive models based on either ETC or NTC predictor set showed comparable classification performance. CONCLUSION: Increased blood cfDNA concentration in late UICC stages inversely correlate with nuclear cfDNA integrity index and suggest that necrotic degradation is not a major cause for higher total cfDNA quantity. The diagnostic and prognostic value of MTCO3 is highly significant in early stages of CRC and can be evaluated more comprehensively, using ETC for qPCR analysis. TRIAL REGISTRATION: The study was registered retrospectively on DRKS, the german register for clinical trials (DRKS00030257, 29/09/2022).

Anti-cancer agent 3-bromopyruvate reduces growth of MPNST and inhibits metabolic pathways in a representative in-vitro model.

BACKGROUND: Anticancer compound 3-bromopyruvate (3-BrPA) suppresses cancer cell growth via targeting glycolytic and mitochondrial metabolism. The malignant peripheral nerve sheath tumor (MPNST), a very aggressive, therapy resistant, and Neurofibromatosis type 1 associated neoplasia, shows a high metabolic activity and affected patients may therefore benefit from 3-BrPA treatment. To elucidate the specific mode of action, we used a controlled cell model overexpressing proteasome activator (PA) 28, subsequently leading to p53 inactivation and oncogenic transformation and therefore reproducing an important pathway in MPNST and overall tumor pathogenesis. METHODS: Viability of MPNST cell lines S462, NSF1, and T265 in response to increasing doses (0-120 µM) of 3-BrPA was analyzed by CellTiter-Blue® assay. Additionally, we investigated viability, reactive oxygen species (ROS) production (dihydroethidium assay), nicotinamide adenine dinucleotide dehydrogenase activity (NADH-TR assay) and lactate production (lactate assay) in mouse B8 fibroblasts overexpressing PA28 in response to 3-BrPA application. For all experiments normal and nutrient deficient conditions were tested. MPNST cell lines were furthermore characterized immunohistochemically for Ki67, p53, bcl2, bcl6, cyclin D1, and p21. RESULTS: MPNST significantly responded dose dependent to 3-BrPA application, whereby S462 cells were most responsive. Human control cells showed a reduced sensitivity. In PA28 overexpressing cancer cell model 3-BrPA application harmed mitochondrial NADH dehydrogenase activity mildly and significantly failed to inhibit lactate production. PA28 overexpression was associated with a functional glycolysis as well as a partial resistance to stress provoked by nutrient deprivation. 3-BrPA treatment was not associated with an increase of ROS. Starvation sensitized MPNST to treatment. CONCLUSIONS: Aggressive MPNST cells are sensitive to 3-BrPA therapy in-vitro with and without starvation. In a PA28 overexpression cancer cell model leading to p53 inactivation, thereby reflecting a key molecular feature in human NF1 associated MPNST, known functions of 3-BrPA to block mitochondrial activity and glycolysis were reproduced, however oncogenic cells displayed a partial resistance. To conclude, 3-BrPA was sufficient to reduce NF1 associated MPNST viability potentially due inhibition of glycolysis which should lead to the initiation of further studies and promises a potential benefit for NF1 patients.

Yeast XRS2 and human NBN gene: Experimental evidence for homology using codon optimized cDNA.

The genes, XRS2 in Saccharomyces cerevisiae and NBN in mammals, have little sequence identity at the amino acid level. Nevertheless, they are both found together with MRE11 and RAD50 in a highly conserved protein complex which functions in the repair of DNA double-strand breaks. Here, we have examined the evolutionary and functional relationship of these two genes by cross-complementation experiments. These experiments necessitated sequence correction for specific codon usage before they could be successfully conducted. We present evidence that despite extreme sequence divergence nibrin can, at least partially, replace Xrs2 in the cellular DNA damage response, and Xrs2 is able to promote nuclear localization of MRE11 in NBS cells. We discuss that the extreme sequence divergence reflects a unique adaptive pressure during evolution related to the specific eukaryotic role for both Xrs2 and nibrin in the subcellular localisation of the DNA repair complex. This, we suggest, is of particular relevance when cells are infected by viruses. The conflict hypothesis of co-evolution of DNA repair genes and DNA viruses may thus explain the very low sequence identity of these two homologous genes.

A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle.

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.

Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

Fkh1 and Fkh2 associate with Sir2 to control CLB2 transcription under normal and oxidative stress conditions.

The Forkhead (Fkh) box family of transcription factors is evolutionary conserved from yeast to higher eukaryotes and its members are involved in many physiological processes including metabolism, DNA repair, cell cycle, stress resistance, apoptosis, and aging. In budding yeast, four Fkh transcription factors were identified, namely Fkh1, Fkh2, Fhl1, and Hcm1, which are implicated in chromatin silencing, cell cycle regulation, and stress response. These factors impinge transcriptional regulation during cell cycle progression, and histone deacetylases (HDACs) play an essential role in this process, e.g., the nuclear localization of Hcm1 depends on Sir2 activity, whereas Sin3/Rpd3 silence cell cycle specific gene transcription in G2/M phase. However, a direct involvement of Sir2 in Fkh1/Fkh2-dependent regulation of target genes is at present unknown. Here, we show that Fkh1 and Fkh2 associate with Sir2 in G1 and M phase, and that Fkh1/Fkh2-mediated activation of reporter genes is antagonized by Sir2. We further report that Sir2 overexpression strongly affects cell growth in an Fkh1/Fkh2-dependent manner. In addition, Sir2 regulates the expression of the mitotic cyclin Clb2 through Fkh1/Fkh2-mediated binding to the CLB2 promoter in G1 and M phase. We finally demonstrate that Sir2 is also enriched at the CLB2 promoter under stress conditions, and that the nuclear localization of Sir2 is dependent on Fkh1 and Fkh2. Taken together, our results show a functional interplay between Fkh1/Fkh2 and Sir2 suggesting a novel mechanism of cell cycle repression. Thus, in budding yeast, not only the regulation of G2/M gene expression but also the protective response against stress could be directly coordinated by Fkh1 and Fkh2.

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes.

The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205 kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P2(1) and P2(1)2(1)2(1) in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52-357 of Epf. Complete data sets were collected to 2.0 and 1.6 Šresolution, respectively, at the Australian Synchrotron.

Sic1 plays a role in timing and oscillatory behaviour of B-type cyclins.

Budding yeast cell cycle oscillates between states of low and high cyclin-dependent kinase activity, driven by association of Cdk1 with B-type (Clb) cyclins. Various Cdk1-Clb complexes are activated and inactivated in a fixed, temporally regulated sequence, inducing the behaviour known as "waves of cyclins". The transition from low to high Clb activity is triggered by degradation of Sic1, the inhibitor of Cdk1-Clb complexes, at the entry to S phase. The G(1) phase is characterized by low Clb activity and high Sic1 levels. High Clb activity and Sic1 proteolysis are found from the beginning of the S phase until the end of mitosis. The mechanism regulating the appearance on schedule of Cdk1-Clb complexes is currently unknown. Here, we analyse oscillations of Clbs, focusing on the role of their inhibitor Sic1. We compare mathematical networks differing in interactions that Sic1 may establish with Cdk1-Clb complexes. Our analysis suggests that the wave-like cyclins pattern derives from the binding of Sic1 to all Clb pairs rather than from Clb degradation. These predictions are experimentally validated, showing that Sic1 indeed interacts and coexists in time with Clbs. Intriguingly, a sic1Δ strain looses cell cycle-regulated periodicity of Clbs, which is observed in the wild type, whether a SIC1-0P strain delays the formation of Clb waves. Our results highlight an additional role for Sic1 in regulating Cdk1-Clb complexes, coordinating their appearance.

Crystal structure of the minor pilin FctB reveals determinants of Group A streptococcal pilus anchoring.

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-A resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.

Purification, crystallization and preliminary crystallographic analysis of the minor pilin FctB from Streptococcus pyogenes.

The minor pilin FctB is an integral part of the pilus assembly expressed by Streptococcus pyogenes. Since it is located at the cell wall, it can be hypothesized that it functions as a cell-wall anchor for the streptococcal pilus. In order to elucidate its structure, the genes for FctB from the S. pyogenes strains 90/306S and SF370 were cloned for overexpression in Escherichia coli. FctB from strain 90/306S was crystallized by the sitting-drop vapour-diffusion method using sodium citrate as a precipitant. The hexagonal FctB crystals belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 95.15, c = 100.25 A, and diffracted to 2.9 A resolution.

The effect of micrococcal nuclease digestion on nucleosome positioning data.

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.

The laminin-binding protein Lbp from Streptococcus pyogenes is a zinc receptor.

The common pathogen Streptococcus pyogenes colonizes the human skin and tonsils and can invade underlying tissues. This requires the adhesion of S. pyogenes to host surface receptors mediated through adhesins. The laminin-binding protein Lbp has been suggested as an adhesin, specific for the human extracellular matrix protein laminin. Sequence alignments, however, indicate a relationship between Lbp and a family of bacterial metal-binding receptors. To further analyze the role of Lbp in S. pyogenes and its potential role in pathogenicity, Lbp has been crystallized, and its structure has been solved at a resolution of 2.45 A (R = 0.186; R(free) = 0.251). Lbp has the typical metal-binding receptor fold, comprising two globular (beta/alpha)(4) domains connected by a helical backbone. The two domains enclose the metal-binding site, which contains a zinc ion. The interaction of Lbp with laminin was further investigated and shown to be specific in vitro. Localization studies with antibodies specific for Lbp show that the protein is attached to the membrane. The data suggest that Lbp is primarily a zinc-binding protein, and we suggest that its interaction with laminin in vivo may be mediated via zinc bound to laminin.

Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp.

The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 A, beta = 106.27 degrees, and diffracted to 2.5 A resolution.

Hip, an HP1-interacting protein, is a haplo- and triplo-suppressor of position effect variegation.

The Drosophila heterochromatin protein 1 (HP1) regulates epigenetic gene silencing and heterochromatin formation by promoting and maintaining chromatin condensation. Here we report the identification and characterization of an HP1-interacting protein (Hip). Hip interacts with HP1 in vitro and is associated with HP1 in vivo. This interaction is mediated by at least three independent but similar HP1-binding modules of the Hip protein. Hip and HP1 completely colocalize in the pericentric heterochromatin, and both haplo- and triplo-dosage mutations act as dominant suppressors of position effect variegation. These findings identify a player in heterochromatinization and suggest that Hip cooperates with HP1 in chromatin remodeling and gene silencing.