Carbon dots fluorescence can be used to distinguish isobaric peptide and to monitor protein oligomerization dynamics.

Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.

Time-of-flight SIMS investigation of peptides containing cell penetrating sequences.

Surface functionalization with biological molecules, such as peptides or proteins, is a very promising method for developing new biomaterials with many potential applications. However, due to their chemical complexity, the characterization of biological materials is often a very challenging task. In this context, time-of-flight secondary ion mass spectrometry is a very helpful characterization tool due to its ability to provide very detailed spatially resolved chemical information of the topmost layer. The peculiar emission/ion formation mechanisms involved in ToF-SIMS analysis often do not allow the detection of the molecular ion of proteins and peptides, providing a rich fragmentation pattern, which is difficult to be related to the surface composition using a univariate approach, due to the relevant number of peaks in the SIMS spectra of peptides and proteins and the slight differences in intensities between different samples. Therefore, we used multivariate analysis to extract the information contained in the ToF-SIMS spectra of four peptides with high amino acid sequence similarity along the peptide chain. The reference peptide (TAT1) is a 12-unit sequence of six amino acids (GRKKRRQRRRPS). The other three peptides have been obtained by inserting a bAla-H dipeptide (carnosine) in three different positions inside the TAT1 chain, namely, GRKKRRQRRRPS-bAla-H (TAT1-Car), bAla-HGRKKRRQRRRPS (Car-TAT1), and GRKKRRQ-bAla-H-RRRPS (T-Car-T). We show that these peptides can be distinguished by ToF-SIMS combined with multivariate data analysis.

Insights into the binding of Ag ions with SilE model peptides: an NMR and MS coupled approach.

The diffuse and renewed use of silver as antimicrobial agent has caused the development of resistance to silver ions in some bacterial strains, posing a serious threat for health systems. In order to cast light on the mechanistic features of resistance, here, we aimed to understand how silver interacts with the periplasmic metal-binding protein SilE which is engaged in bacterial silver detoxification. This aim was addressed by studying two peptide portions of SilE sequence (SP2 and SP3) that contain the putative motifs involved in Ag+ binding. We demonstrate that SP2 model peptide is involved in silver binding through its histidine and methionine residues in the two HXXM binding sites. In particular, the first binding site is supposed to bind the Ag+ ion in a linear fashion, while the second binding site complexes the silver ion in a distorted trigonal planar fashion. We propose a model where the SP2 peptide binds two silver ions when the concentration ratio Ag+/SP2 is ≥10.0. We also suggest that the two binding sites of SP2 have different affinity for silver. This evidence comes from the change in the path direction of the Nuclear Magnetic Resonance (NMR) cross-peaks upon the addition of Ag+. Here, we report the conformational changes of SilE model peptides occurring upon silver binding, monitored at a deep level of molecular details. This was addressed by a multifaceted approach, combining NMR, circular dichroism, and mass spectrometry experiments.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Dipyridamole for tracking amyloidogenic proteins aggregation and enhancing polyubiquitination.

Dipyridamole is currently used as a medication that inhibits blood clot formation and it is also investigated in the context of neurodegenerative and other amyloid related diseases. Here, we propose this molecule as a new diagnostic tool to follow the aggregation properties of three different amyloidogenic proteins tested (insulin, amylin and amyloid ß peptide 1-40). Results show that dipyridamole is sensitive to early stage amyloid formation undetected by thioflavin T, giving a different response for the aggregation of the three different proteins. In addition, we show that dipyridamole is also able to enhance ubiquitin chain growth, paving the way to its potential application as therapeutic agent in neurodegenerative diseases.

Carbon dots surface chemistry drives fluorescent properties: New tools to distinguish isobaric peptides.

The possibility to design rational carbon dots surface functionalization for specific analytical and bioanalytical applications is hindered by the lack of a full knowledge of the surface chemical features driving fluorescent properties. In this model study, we have synthesized four different peptides, three of which are isobaric and not distinguishable by common MSMS experiments. After having characterized the peptides conformations by CD analyses, we have covalently bonded all four peptides to carbon dots by using different experimental procedures, which produce different functional groups on the carbon dots surface. The peptide orientations obtained on the differently functionalized surface of the nanoparticles were different and produced different fluorescent responses. The reported results indicate the possibility to design amino and carboxyl enriched surface carbon dots to answer specific chemical requirements, paving the way for the use of these nanoparticles as a versatile and useful new chemical and biochemical tool.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Histological and Histomorphometric Evaluation of New Bone Formation after Maxillary Sinus Augmentation with Two Different Osteoconductive Materials: A Randomized, Parallel, Double-Blind Clinical Trial.

This study aimed to investigate the histological features of deproteinized equine bone mineral (DEBM) and anorganic bovine bone (ABB) after human sinus augmentation with the lateral approach. Twenty-three sinus augmentations were performed in 16 patients (male: 10/female: 6) using DEBM or ABB in a randomized fashion. Healing took place over the next 6 months. Bone core biopsies (N = 23) were obtained for each subject prior to placing the dental implants. The biopsies were processed for both histological descriptions and histomorphometric analysis. Statistical analyses were applied as appropriate, defining statistical significance as p < 0.05. Core bone biopsies revealed no differences in terms of newly formed bone between groups, or differences in terms of tissue inflammation. Both DEBM and ABB appear to be suitable biomaterials for bone augmentation in sinus lift surgery in the short term. However, dedicated studies are required to confirm these results and their stability in the long term.

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from ß-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism.

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified.

The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas.

Defective DNA damage response (DDR) is frequently associated with tumorigenesis. Abrogation of DDR leads to genomic instability, which is one of the most common characteristics of human cancers. TP53 mutations with gain-of-function activity are associated with tumors under high replicative stress, high genomic instability, and reduced patient survival. The BRCA1 and RAD17 genes encode two pivotal DNA repair proteins required for proper cell-cycle regulation and maintenance of genomic stability. We initially evaluated whether miR-205-5p, a microRNA (miRNA) highly expressed in head and neck squamous cell carcinoma (HNSCC), targeted BRCA1 and RAD17 expression. We found that, in vitro and in vivo, BRCA1 and RAD17 are targets of miR-205-5p in HNSCC, leading to inefficient DNA repair and increased chromosomal instability. Conversely, miR-205-5p downregulation increased BRCA1 and RAD17 messenger RNA (mRNA) levels, leading to a reduction in in vivo tumor growth. Interestingly, miR-205-5p expression was significantly anti-correlated with BRCA1 and RAD17 targets. Furthermore, we documented that miR-205-5p expression was higher in tumoral and peritumoral HNSCC tissues than non-tumoral tissues in patients exhibiting reduced local recurrence-free survival. Collectively, these findings unveil miR-205-5p's notable role in determining genomic instability in HNSCC through its selective targeting of BRCA1 and RAD17 gene expression. High miR-205-5p levels in the peritumoral tissues might be relevant for the early detection of minimal residual disease and pre-cancer molecular alterations involved in tumor development.

Dropwort-induced metabolic reprogramming restrains YAP/TAZ/TEAD oncogenic axis in mesothelioma.

BACKGROUND: Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway. METHODS: The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in "in vitro" and "in vivo" models of MPM. At the molecular level, two "omic" approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis. RESULTS: We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination. CONCLUSIONS: Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management.

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S.

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.

Carnosine protects pancreatic beta cells and islets against oxidative stress damage.

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure.

Surface tailoring of polyacrylate-grafted graphene oxide for controlled interactions at the biointerface.

The actual surface termination and lateral size of a nanomaterial is crucial in its interaction with biomolecules at the aqueous interface. Graphene oxide (GO) nanosheets have been demonstrated as promising nanoplatform for both diagnostic and therapeutic applications. To this respect, 'smart' GO nanocarriers have been obtained by the surface functionalisation with polymers sensitive, e.g., to pH, as the polyacrylate (PAA) case. In this work, hybrid GO/PAA samples prepared respectively at low (GOPAAthin) or high (GOPAAthick) monomer grafting ratio, were scrutinised both theoretically, by molecular dynamic calculations, and experimentally by a multitechnique approach, including spectroscopic (UV-visible, fluorescence, Raman, Attenuated-total reflectance-Fourier transformed infrared and X-ray photoelectron spectroscopies), spectrometric (time-of-flight secondary ion and electrospray ionisation mass spectrometries) and microscopic (atomic force and confocal microscopies) methods. The actual surface termination, evaluated in terms of the relative ratio between polar and dispersive groups at the surface of the GO/polymer systems, was found to correlate with the average orientation of hydrophilic/hydrophobic domains of albumin, used as model protein. Moreover, the comparison among GO, GO-PAAthin and GO-PAAthick in the optical response at the interface with aqueous solutions, both at acid and at physiological pH, showed that the hybrid GO-polymer platform could be suitable not only to exploit a pH-triggered drug release but also for a modulation of the GO intrinsic emission properties. Energy transfer experiments on the GO/polymer oxide/fluorescein-labelled albumin/doxorubicin assembly showed significant differences for GO and GO-PAA samples, thus demonstrating the occurrence of different electronic processes at the hybrid nano-bio-interfaces. Confocal microscopy studies of cellular uptake in neuroblastoma cells confirmed the promising potentialities of the developed nanoplatform for applications at the biointerface.

Multiple functions of insulin-degrading enzyme: a metabolic crosslight?

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as ß-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the "aggregopathies" and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin-proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.

AMPK ß1 reduces tumor progression and improves survival in p53 null mice.

The AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK ß1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK ß1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK ß1 accelerates the appearance of a T-cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin-1ß (IL1ß), reductions in acetyl-CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK ß1 subunit accelerate the development of T-cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53-mutant tumors.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.

Monitoring the biomolecular interactions and the activity of Zn-containing enzymes involved in conformational diseases: experimental methods for therapeutic purposes.

Zinc metalloproteases (ZnMPs) participate in diverse biological reactions, encompassing the synthesis and degradation of all the major metabolites in living organisms. In particular, ZnMPs have been recognized to play a very important role in controlling the concentration level of several peptides and/or proteins whose homeostasis has to be finely regulated for the correct physiology of cells. Dyshomeostasis of aggregation-prone proteins causes pathological conditions and the development of several different diseases. For this reason, in recent years, many analytical approaches have been applied for studying the interaction between ZnMPs and their substrates/inhibitors and how environmental factors can affect enzyme activities. In this scenario, nuclear magnetic resonance, X-ray diffraction, mass spectrometric (MS), and optical methods occupy a very important role in elucidating different aspects of the ZnMPs-substrates/inhibitors interaction, ranging from identification of cleavage sites to quantitation of kinetic parameters and inhibition constants. Here, an overview of all the main achievements in the application of different experimental approaches with special attention to MS methods to the investigation of ZnMPs-substrates/inhibitors interaction is given. A general MS experimental protocol which has been proved to be useful to study such interactions is also described.

The role of copper(II) in the aggregation of human amylin.

Amylin is a 37-residue peptide hormone produced by the islet ß-cells of pancreas and the formation of amylin aggregates is strongly associated with ß-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aß and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.