Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for γ-secretase activity.

Fn14 is a cell surface receptor with key functions in tissue homeostasis and injury but is also linked to chronic diseases. Despite its physiological and medical importance, the regulation of Fn14 signaling and turnover is only partly understood. Here, we demonstrate that Fn14 is cleaved within its transmembrane domain by the protease γ-secretase, resulting in secretion of the soluble Fn14 ectodomain (sFn14). Inhibition of γ-secretase in tumor cells reduced sFn14 secretion, increased full-length Fn14 at the cell surface, and enhanced TWEAK ligand-stimulated Fn14 signaling through the NFκB pathway, which led to enhanced release of the cytokine tumor necrosis factor. γ-Secretase-dependent sFn14 release was also detected ex vivo in primary tumor cells from glioblastoma patients, in mouse and human plasma and was strongly reduced in blood from human cancer patients dosed with a γ-secretase inhibitor prior to chimeric antigen receptor (CAR)-T-cell treatment. Taken together, our study demonstrates a novel function for γ-secretase in attenuating TWEAK/Fn14 signaling and suggests the use of sFn14 as an easily measurable pharmacodynamic biomarker to monitor γ-secretase activity in vivo.

ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data.

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI-IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI-IMS. This is achieved by combining information from IMS and shotgun proteomics (LC-MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC-MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis.

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM+ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM+ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206+ and peritubular MHC II+ macrophages, with higher levels in CD206+ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2+ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM+ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206+MHC II+ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2+ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM+ mice. The changes in testis are distinct from the described alterations in other organs of AROM+, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM+ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM+ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.

Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice.

Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or Pde6b-mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectroscopy-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify NAcetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.

MALDI-IMS combined with shotgun proteomics identify and localize new factors in male infertility.

Spermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wild-type and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of pro-inflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterize AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples.

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.

Association of a Zn(2+) containing metallo ß-lactamase with the anticancer antibiotic mithramycin.

Pathogenic bacteria that are resistant to ß-lactam antibiotics mostly utilize serine ß-lactamases to degrade the antibiotics. Current studies have shown that different subclasses of metallo ß-lactamases (E[MBL]) are involved in the defense mechanism of drug resistant bacteria. Here we report that the Zn(2+) containing subclass B1 E[MBL] from Bacillus cereus binds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic (CD and fluorescence) and isothermal titration calorimetry studies show that MTR forms a high affinity complex with the Zn(2+) ion containing E[MBL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of this high affinity complex occurs due to the potential of MTR to bind bivalent metal ions like Zn(2+). Furthermore, CD spectroscopy, dynamic light scattering and differential scanning calorimetry studies indicate that the strong association with sub-micromolar dissociation constant leads to an alteration in the enzyme conformation at both secondary and tertiary structural levels. The enzyme activity decreases as a consequence to this conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn(2+) and the enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor.

Association of antitumor antibiotic Mithramycin with Mn2+ and the potential cellular targets of Mithramycin after association with Mn2+.

Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn(2+)). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn(2+) in the mole ratio of 2:1 [MTR: Mn(2+)] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn(2+)] has a right-handed twist conformation similar in structure with the complexes reported for Mg(2+) and Zn(2+). This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn(2+)] complex binds to double-stranded DNA with an apparent dissociation constant of 32 µM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298 µM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn(2+) containing metalloenzyme manganese superoxide dismutase from Escherichia coli.

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic.

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu(2+). CHR forms a high affinity 2:1 (CHR:Cu(2+)) complex with dissociation constant of 0.08 × 10(-10) M(2) at 25°C, pH 8.0. The affinity of CHR for Cu(2+) is higher than those for Mg(2+) and Zn(2+) reported earlier from our laboratory. CHR binds preferentially to Cu(2+) in presence of equimolar amount of Zn(2+). Complex formation between CHR and Cu(2+) is an entropy driven endothermic process. Difference between calorimetric and van't Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)(2):Cu(2+)] complex assumes a structure different from either of the Mg(2+) and Zn(2+) complex reported earlier. Both [(CHR)(2):Mg(2+)] and [(CHR)(2):Zn(2+)] complexes are known to bind DNA. In contrast, [(CHR)(2):Cu(2+)] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5'-CCGGCGCCGG-3'). In order to interact with double helical DNA, the (antibiotic)(2) : metal (Mg(2+) and Zn(2+)) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu(2+) complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg(2+) and Zn(2+). The results also indicate that CHR has a potential for chelation therapy in Cu(2+) accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.

Self-association of the anionic form of the DNA-binding anticancer drug mithramycin.

The aqueous-phase self-association of mithramycin (MTR), an aureolic acid anticancer antibiotic, has been studied using different spectroscopic techniques such as absorption, fluorescence, circular dichroism, and 1H nuclear magnetic resonance spectroscopy. Results from these studies indicate self-association of the anionic antibiotic at pH 8.0 over a concentration range from micromolar to millimolar. These results could be ascribed to the following steps of self-association: M + M left arrow over right arrow M2, M2 + M left arrow over right arrow M3, and M3 + M left arrow over right arrow M4, where M, M2, M3, and M4 represent the monomer, dimer, trimer, and tetramer of mithramycin, respectively. Dynamic light scattering and isothermal titration calorimetry studies also support aggregation. In contrast, an insignificant extent of self-association is found for the neutral drug (at pH 3.5) and the [(MTR)2Mg2+] complex (at pH 8.0). Analysis of 2D NMR spectra of 1 mM MTR suggests that the sugar moieties play a role in the self-association process. Self-association of the drug might occur either via hydrophobic interaction of the sugar residues among themselves or water-mediated hydrogen bond formation between sugar residue(s). On the other hand, absence of a significant upfield shift of the aromatic protons from 100 microM to 1 mM MTR suggests against the possibility of stacking interactions between the aromatic rings as a stabilizing force for the formation of the dimer and higher oligomers.