Interaction of Plectin with Keratins 5 and 14: Dependence on Several Plectin Domains and Keratin Quaternary Structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.Journal of Investigative Dermatology advance online publication, 10 July 2014; doi:10.1038/jid.2014.255.

A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Autologous hematopoietic stem cell transplantation vs intravenous pulse cyclophosphamide in diffuse cutaneous systemic sclerosis: a randomized clinical trial

IMPORTANCE High-dose immunosuppressive therapy and autologous hematopoietic stem cell transplantation (HSCT) have shown efficacy in systemic sclerosis in phase 1 and small phase 2 trials. OBJECTIVE To compare efficacy and safety of HSCT vs 12 successive monthly intravenous pulses of cyclophosphamide. DESIGN, SETTING, AND PARTICIPANTS The Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial, a phase 3, multicenter, randomized (1:1), open-label, parallel-group, clinical trial conducted in 10 countries at 29 centers with access to a European Group for Blood and Marrow Transplantation-registered transplant facility. From March 2001 to October 2009, 156 patients with early diffuse cutaneous systemic sclerosis were recruited and followed up until October 31, 2013. INTERVENTIONS HSCT vs intravenous pulse cyclophosphamide. MAIN OUTCOMES AND MEASURES The primary end point was event-free survival, defined as time from randomization until the occurrence of death or persistent major organ failure. RESULTS A total of 156 patients were randomly assigned to receive HSCT (n = 79) or cyclophosphamide (n = 77). During a median follow-up of 5.8 years, 53 events occurred: 22 in the HSCT group (19 deaths and 3 irreversible organ failures) and 31 in the control group (23 deaths and 8 irreversible organ failures). During the first year, there were more events in the HSCT group (13 events [16.5%], including 8 treatment-related deaths) than in the control group (8 events [10.4%], with no treatment-related deaths). At 2 years, 14 events (17.7%) had occurred cumulatively in the HSCT group vs 14 events (18.2%) in the control group; at 4 years, 15 events (19%) had occurred cumulatively in the HSCT group vs 20 events (26%) in the control group. Time-varying hazard ratios (modeled with treatment × time interaction) for event-free survival were 0.35 (95% CI, 0.16-0.74) at 2 years and 0.34 (95% CI, 0.16-0.74) at 4 years. CONCLUSIONS AND RELEVANCE Among patients with early diffuse cutaneous systemic sclerosis, HSCT was associated with increased treatment-related mortality in the first year after treatment. However, HCST conferred a significant long-term event-free survival benefit. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN54371254.

Quantification of nanoparticles at the single-cell level: an overview about state-of-the-art techniques and their limitations.

With the increasing production and use of engineered nanoparticles it is crucial that their interaction with biological systems is understood. Due to the small size of nanoparticles, their identification and localization within single cells is extremely challenging. Therefore, various cutting-edge techniques are required to detect and to quantify metals, metal oxides, magnetic, fluorescent, as well as electron-dense nanoparticles. Several techniques will be discussed in detail, such as inductively coupled plasma atomic emission spectroscopy, flow cytometry, laser scanning microscopy combined with digital image restoration, as well as quantitative analysis by means of stereology on transmission electron microscopy images. An overview will be given regarding the advantages of those visualization/quantification systems, including a thorough discussion about limitations and pitfalls.

Interaction of small molecules with specific immune receptors: the p-i concept and its consequences

Drugs may stimulate the immune system by forming stable new antigenic complexes consisting of the drug or drug metabolite which is covalently bound to a protein or peptide (hapten-carrier complex). Both, B- and T-cell immunity may arise, the latter directed to hapten modified peptides presented by HLA molecules. Beside this immunological stimulation, drugs can also stimulate the immune system through binding by non-covalent bonds to proteins like immune receptors. This so-called “pharmacological interaction with immune receptors” concept (“p-i concept”) may occur with HLA or TCR molecules themselves (p-i HLA or p-i TCR), and not the immunogenic peptide. It is a type of “off-target” activity of the drug on immune receptors, but more complex as various cell types, cell interactions and functionally different T cells are involved. In this review the conditions which lead to activation of T cells by p-i are discussed: important factors for a functional consequence of drug binding is the location of binding (p-i HLA or p-i TCR); the exact site within these immune receptors; the affinity of binding and the finding that p-i HLA can stimulate the immune system like an allo-allele. The p-i concept is able to solve some puzzles of drug hypersensitivity reactions and are a basis to better treat and potentially avoid drug hypersensitivity reactions. Moreover, the p-i concept shows that in contrast to previous beliefs small molecules do interact with immune receptors with functional consequence. But these interactions are not based on “immune recognition”, are at odds with some immunological concepts, but may nevertheless open new possibilities to understand and even treat immune reactions

PSGL-1 and E/P-selectins are essential for T-cell rolling in inflamed CNS microvessels but dispensable for initiation of EAE

T-cell migration across the blood-brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P-selectin glycoprotein ligand-1 (PSGL-1) and its endothelial ligands E- and P-selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL-1 or E/P-selectins does not result in ameliorated EAE, we speculated that T-cell entry into the spinal cord is independent of PSGL-1 and E/P-selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL-1(-/-) proteolipid protein-specific T cells in inflamed spinal cord microvessels of WT or E/P-selectin(-/-) SJL/J mice during EAE. T-cell rolling but not T-cell capture was completely abrogated in the absence of either PSGL-1 or E- and P-selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T-cell invasion into the CNS parenchyma. Thus, PSGL-1 interaction with E/P-selectin is essential for T-cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T-cell rolling is not required for successful T-cell entry into the CNS and initiation of EAE.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118.

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression.

Systemic root signalling in a belowground, volatile-mediated tritrophic interaction

Plants attacked by leaf herbivores release volatile organic compounds (VOCs) both locally from the wounded site and systemically from non-attacked tissues. These volatiles serve as attractants for predators and parasitoids. This phenomenon is well described for plant leaves, but systemic induction of VOCs in the roots has remained unstudied. We assessed the spatial and temporal activation of the synthesis and release of (E)-β-caryophyllene (EβC) in maize roots upon feeding by larvae of Diabrotica virgifera virgifera, as well as the importance of systemically produced EβC for the attraction of the entomopathogenic nematode Heterorhabditis megidis. The production of EβC was found to be significantly stronger at the site of attack than in non-attacked tissues. A weak, but significant, increase in transcriptional activity of the EβC synthase gene tps23 and a corresponding increase in EβC content were observed in the roots above the feeding site and in adjacent roots, demonstrating for the first time that herbivory triggers systemic production of a volatile within root systems. In belowground olfactometers, the nematodes were significantly more attracted towards local feeding sites than systemically induced roots. The possible advantages and disadvantages of systemic volatile signalling in roots are discussed.

Synthesis and Structural Characterizations of New Coordination Polymers Generated by the Interaction Between the Trinuclear Triangular SBU [Cu 3 (μ 3 -OH)(μ-pz) 3 ] 2+ and 4,4′-Bipyridine. 3°

The reactions of 4,4′-bipyridine with selected trinuclear triangular copper(II) complexes, [Cu3(μ3-OH)(μ-pz)3(RCOO)2Lx], [pz = pyrazolate anion, R = CH3(CH2)n (2 ≤ n ≤ 5); L = H2O, MeOH, EtOH] yielded a series of 1D coordination polymers (CPs) based on the repetition of [Cu3(μ3-OH)(μ-pz)3] secondary building units joined by bipyridine. The CPs were characterized by conventional analytical methods (elemental analyses, ESI-MS, IR spectra) and single crystal XRD determinations. An unprecedented 1D CP, generated through the bipyridine bridging hexanuclear copper clusters moieties, two 1D CPs presenting structural analogies, and two monodimensional tapes having almost exactly superimposable structures, were obtained. In one case, the crystal packing makes evident the presence of small, not-connected pores, accounting for ca. 6% of free cell volume.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

The Gas6-Axl Interaction Mediates Endothelial Uptake of Platelet Microparticles.

Upon activation, platelets release plasma-membrane derived microparticles (PMPs) exposing phosphatidylserine (PS) on their surface. The function and clearance mechanism of these MPs are incompletely understood. As they are pro-coagulant and potentially pro-inflammatory, rapid clearance from the circulation is essential for prevention of thrombotic diseases. The tyrosine kinase receptors Tyro3, Axl and Mer (TAMs) and their ligands protein S and Gas6 are involved in the uptake of PS-exposing apoptotic cells in macrophages and dendritic cells. Both TAMs and their ligands are expressed in the vasculature, the functional significance of which is poorly understood. In this study we investigated how vascular TAMs and their ligands may mediate endothelial uptake of PMPs. PMPs, generated from purified human platelets, were isolated by ultracentrifugation and labeled with biotin or PKH67. The uptake of labeled MPs in the presence of protein S and Gas6 in human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) was monitored by flow cytometry, western blotting and confocal/electron microscopy. We found that both endothelial cell types can phagocytose PMPs, and using TAM-blocking antibodies or siRNA knock-down of individual TAMs we show that the uptake is mediated by endothelial Axl and Gas6. As circulating PMPs-levels were not altered in Gas6-/- mice compared to Gas6+/+ mice, we hypothesize that the Gas6-mediated uptake is not a means to clear the bulk of circulating PMPs but may serve to phagocytose PMPs locally generated at sites of platelet activation and as a way to affect endothelial responses.