The primary cilium (PC) was considered as a vestigial organelle with no significant physiological importance, until the discovery that PC perturbation disturbs several signalling pathways and results in the dysfunction of a variety of organs. Genetic studies have demonstrated that mutations affecting PC proteins or its anchoring structure, the basal body, underlie a class of human disorders (known as ciliopathies) characterized by a constellation of clinical signs. Further investigations have demonstrated that the PC is involved in a broad range of biological processes, in both developing and mature tissues. Kidney disease is a common clinical feature of cilia disorders, supporting the hypothesis of a crucial role of the PC in kidney homoeostasis. Clinical proteomics and metabolomics are an expanding research area. Interestingly, the application of these methodologies to the analysis of urine, a biological sample that can be collected in a non-invasive fashion and possibly in large amounts, makes these studies feasible also in patients. The present article describes the most recent proteomic and metabolomic studies exploring kidney dysfunction in the setting of ciliopathies, showing the potential of these methodologies in the elucidation of disease pathophysiology and in the discovery of biomarkers.
The G-quadruplex-forming telomeric sequence (TTAGGG)4TT was investigated by polarized Ultraviolet Resonance Raman Scattering (UVRR) at 266 nm. The presence of 40% poly(ethylene glycol) and the so-called "self-crowding" condition were used to induce the hybrid-to-parallel topology transition. Analysis of frequency shifts with temperature showed the role of several functional groups in the topological transitions and provides structural dynamical information. Circular dichroism under similar conditions was used as a reference. UVRR shed light on the effect of intramolecular interactions and of local and environmental dynamics in promoting different G-quadruplex topologies, induced by solution conditions or by temperature changes. Overall, these findings showed the enormous potential of this spectroscopy for G-quadruplex conformational studies.
The Ru-based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, Ru and Ru, was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent. To better understand this process, a pH-dependent, spectroelectrochemical surface-enhanced Raman scattering (SERS) study was performed also on AziRu-functionalized Au electrodes as a surrogate and simplest model system of Ru- and Ru-based drugs. This SERS study provided a p K of 6.0 ± 0.4 for aquated AziRu in the Ru state, which falls in the watershed range of pH values separating most cancer environments from their physiological counterparts. These experiments also indicate a dramatic shift of the redox potential E by >600 mV of aquated AziRu toward more positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known Ru complex NAMI-A) will modulate the p K and E, without affecting the underlying reaction mechanism.
Up-regulated Gene clone 7 (URG7) is an ER resident protein, whose expression is upregulated in the presence of hepatitis B virus X antigen (HBxAg) during HBV infection. In virus-infected hepatocytes, URG7 shows an anti-apoptotic activity due to the PI3K/AKT signaling activation, does not seem to have tumorigenic properties, but it appears to promote the development and progression of fibrosis. However, the molecular mechanisms underlying URG7 activity remain largely unknown.
Huntington's disease is a dreadful, incurable disorder. It springs from the autosomal dominant mutation in the first exon of the HTT gene, which encodes for the huntingtin protein (HTT) and results in progressive neurodegeneration. Thus far, all the attempted approaches to tackle the mutant HTT-induced toxicity causing this disease have failed. The mutant protein comes with the aberrantly expanded poly-glutamine tract. It is primarily to blame for the build-up of β-amyloid-like HTT aggregates, deleterious once broadened beyond the critical ∼35-37 repeats threshold. Recent experimental findings have provided valuable information on the molecular basis underlying this HTT-driven neurodegeneration. These findings indicate that the poly-glutamine siding regions and many post-translation modifications either abet or counter the poly-glutamine tract. This review provides an overall, up-to-date insight into HTT biophysics and structural biology, particularly discussing novel pharmacological options to specifically target the mutated protein and thus inhibit its functions and toxicity.
The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.
Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC)·poly(dG-dC) and the poly(dA-dT)·poly(dA-dT), by means of UV-Vis and circular dichroism (CD) spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues.
The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
A combination of mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography has been used to obtain detailed information on the reaction of an iridium-based CO-releasing molecule (Ir-CORM), Cs2IrCl5CO, with a model protein, bovine pancreatic ribonuclease. The results show that Ir-compound fragments bind to the N-terminal amine and close to histidine and methionine side chains, and the CO ligand is retained for a long time. The data provide helpful information for identifying protein targets for Ir-CORMs and for studying the mechanism that allows them to exhibit their interesting biological properties.
Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m(2), respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.