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.

Surface Plasmon Resonance Allows to Correlate Molecular Properties With Diffusion Coefficients of Linear Chain Alcohols.

Every biological and physicochemical process occurring in a fluid phase depends on the diffusion coefficient (D) of the species in solution. In the present work, a model to describe and fit the behaviour of D ${D}$ as a function of structure and extensive thermodynamics parameters in binary solutions of linear chain organic molecules is developed. Supporting experimental and computational evidences for this model are obtained by measuring D ${D}$ for a series of n ${n}$ -alcohols through a novel surface plasmon resonance method and molecular dynamics simulations. This allows to propose a kind of combined analysis to explain the dependence of D ${D}$ on various thermodynamic and structural parameters. The results suggest that for small linear systems in the range from 0 to 200 g mol-1 and under the assumption that the diffusive activation energy is a linear function of mass, D ${D}$ is strictly dependent on the molecular shape and on the relative strength of the solute-solvent intermolecular forces represented by a parameter named R. The newly proposed approach can be utilized to characterize and monitor progressive changes in physicochemical properties for any investigated species upon increasing the dimension of the aggregate/molecule along a certain direction.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, ß-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Carbon dots as a versatile tool to monitor insulin aggregation.

The possibility to monitor peptide and protein aggregation is of paramount importance in the so-called conformational diseases, as the understanding of many physiological pathways, as well as pathological processes involved in the development of such diseases, depends very much on the actual possibility to monitor biomolecule oligomeric distribution and aggregation. In this work, we report a novel experimental method to monitor protein aggregation, based on the change of the fluorescent properties of carbon dots upon protein binding. The results obtained in the case of insulin with this newly proposed experimental approach are compared with those obtained with other common experimental techniques normally used for the same purpose (circular dichroism, DLS, PICUP and ThT fluorescence). The greatest advantage of the hereby presented methodology over all the other experimental methods considered is the possibility to monitor the initial stages of insulin aggregation under the different experimental conditions sampled and the absence of possible disturbances and/or molecular probes during the aggregation process.

GxxxG Motif Stabilize Ion-Channel like Pores through Cα-H···O Interaction in Aß (1-40).

Aß (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm-1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

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.

An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer's disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA. Results indicate that both positive and negative modulations of the IDE activity can be correlated to an increase and a decrease of the measured Hill coefficient, respectively, giving a new insight into the IDE activity mechanism. The use of the IDE R767A mutant for which oligomerization is hindered confirmed that the positive allosteric modulation of IDE by carnosine is due to a change in the enzyme oligomeric state occurring also for the enzyme immobilized on the gold SPR chip.

THE USE OF MASS SPECTROMETRY TO STUDY ZN-METALLOPROTEASE-SUBSTRATE INTERACTIONS.

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 and how environmental factors can affect enzyme activities. In this scenario, mass spectrometric methods occupy a very important role in elucidating different aspects of ZnMPs-substrates interaction. These range from identification of cleavage sites to quantitation of kinetic parameters. In this work, an overview of all the main achievements regarding the application of mass spectrometric methods to investigating ZnMPs-substrates interactions is presented. A general experimental protocol is also described which may prove useful to the study of similar interactions. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism.

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.

Mass spectrometry is a multifaceted weapon to be used in the battle against Alzheimer's disease: Amyloid beta peptides and beyond.

Amyloid-ß peptide (Aß) accumulation and aggregation have been considered for many years the main cause of Alzheimer's disease (AD), and therefore have been the principal target of investigation as well as of the proposed therapeutic approaches (Grasso [2011] Mass Spectrom Rev. 30: 347-365). However, the amyloid cascade hypothesis, which considers Aß accumulation the only causative agent of the disease, has proven to be incomplete if not wrong. In recent years, actors such as metal ions, oxidative stress, and other cofactors have been proposed as possible co-agents or, in some cases, main causative factors of AD. In this scenario, MS investigation has proven to be fundamental to design possible diagnostic strategies of this elusive disease, as well as to understand the biomolecular mechanisms involved, in the attempt to find a possible therapeutic solution. We review the current applications of MS in the search for possible Aß biomarkers of AD to help the diagnosis of the disease. Recent examples of the important contributions that MS has given to prove or build theories on the molecular pathways involved with such terrible disease are also reviewed.

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.

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.

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.

Atmospheric pressure MALDI for the noninvasive characterization of carbonaceous ink from Renaissance documents.

The analytical methods that are usually applied to determine the compositions of inks from ancient manuscripts usually focus on inorganic components, as in the case of iron gall ink. In this work, we describe the use of atmospheric pressure/matrix-assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) as a spatially resolved analytical technique for the study of the organic carbonaceous components of inks used in handwritten parts of ancient books for the first time. Large polycyclic aromatic hydrocarbons (L-PAH) were identified in situ in the ink of XVII century handwritten documents. We prove that it is possible to apply MALDI-MS as a suitable microdestructive diagnostic tool for analyzing samples in air at atmospheric pressure, thus simplifying investigations of the organic components of artistic and archaeological objects. The interpretation of the experimental MS results was supported by independent Raman spectroscopic investigations. Graphical abstract Atmospheric pressure/MALDI mass spectrometry detects in situ polycyclic aromatic hydrocarbons in the carbonaceous ink of XVII century manuscripts.

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.

Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

Gender Incongruence: Prevalence and Therapeutic Options Among Transsexuals Belonging to a Single Center.

AIM: This study aimed to describe characteristics and treatment choices among AMABs (Assigned Male At Birth) and AFABs (Assigned Female At Birth) transgenders enrolled from March 2021 to July 2023 at the PTA S. Giorgio of the ASP3-Catania. CASE HISTORY: A total of 145 patients were studied, and there was no prevalence of AMAB/AFAB. At first observation for AMABs, the age was 26 years and 25 years for AFABs, with 11 AMAB/AFAB declared as "non-binary" (average age 17 years). RESULTS: In AMAB/AFAB, we evaluated hormonal treatment, efficacy, and dosage/hormonal levels. In AMABs, oral estradiol valerate (4 mg/day) or transdermal estradiol in gel (2 mg/day) + oral cyproterone acetate (25 mg/day) for both estrogenic formulations were used. Testosterone (TE), LH, FSH, and PRL at baseline and during chronic treatment were measured. In AFABs, we used injectable TE (250 mg/3-4 weeks or 1 g/12-16 weeks) or transdermal TE (60- 80 mg/day). In these patients, we analyzed blood count, LH, FSH, and TE. Hematocrit, hemoglobin, and red blood cell count showed a modest elevation after 4-6 months of treatment. About 32% of AFABs complained of transient uterine bleeding, but no hypertension or ovarian pathology was detected. CONCLUSION: In AMABs, despite the short observation period, no patient showed an increased risk of myocardial infarction and ischemic stroke. Among AFABs, no increased risk of cardiovascular or cerebrovascular disease was observed. Furthermore, given the complexity of the phenomenon, the integration between the different professional figures who require specific and qualified skills is fundamental.

Detection of insulin oligomeric forms by a novel surface plasmon resonance-diffusion coefficient based approach.

Insulin is commonly used to treat diabetes and undergoes aggregation at the site of repeated injections in diabetic patients. Moreover, aggregation is also observed during its industrial production and transport and should be avoided to preserve its bioavailability to correctly adjust glucose levels in diabetic patients. However, monitoring the effect of various parameters (pH, protein concentration, metal ions, etc.) on the insulin aggregation and oligomerization state is very challenging. In this work, we have applied a novel Surface Plasmon Resonance (SPR)-based experimental approach to insulin solutions at various experimental conditions, monitoring how its diffusion coefficient is affected by pH and the presence of metal ions (copper and zinc) with unprecedented sensitivity, precision, and reproducibility. The reported SPR method, hereby applied to a protein for the first time, besides giving insight into the insulin oligomerization and aggregation phenomena, proved to be very robust for determining the diffusion coefficient of any biomolecule. A theoretical background is given together with the software description, specially designed to fit the experimental data. This new way of applying SPR represents an innovation in the bio-sensing field and expanding the potentiality of commonly used SPR instruments well over the canonical investigation of biomolecular interactions.

Blockage of melatonin receptors impairs p53-mediated prevention of DNA damage accumulation.

Melatonin has been known to be a chemopreventive agent since its levels inversely correlate with the risk of developing cancer. We have recently shown that melatonin induces p38-dependent phosphorylation of both p53 and histone H2AX. This is associated with a p53-mediated increase in repair of both endogenous and chemotherapy-induced DNA damage. In addition, the inhibition of p38 activities impairs melatonin's capability to induce a p53-dependent DNA damage response and thus its ability to maintain genome integrity. Since melatonin-induced p53 phosphorylation requires an intact p38 phosphorylation cascade and p38 can be activated by G proteins, we supposed that melatonin's activities could be mediated by its G-protein-coupled membrane receptors, MT1 and MT2. Here, we show that the activation of the p53-dependent DNA damage response by melatonin is indeed mediated by MT1 and MT2. As a result, the absence of either receptor impairs melatonin's ability to reduce both cell proliferation and clonogenic potential of cancer cells. In addition, this causes an impairment of the p53-dependent DNA damage response. By providing molecular insight, our findings might have translational impact, suggesting the involvement of melatonin receptors in tumorigenesis.

Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.