Search

ATP has recently been reconsidered as a molecule with functional properties which go beyond its recognized role of the energetic driver of the cell. ATP has been described as an allosteric modulator as well as a biological hydrotrope with anti-aggregation properties in the crowded cellular environment. The role of ATP as a modulator of the homeostasis of the neurotrophins (NTs), a growth factor protein family whose most known member is the nerve growth factor (NGF), has been investigated. The modulation of NTs by small endogenous ligands is still a scarcely described area, with few papers reporting on the topic, and very few reports on the molecular determinants of these interactions. However, a detailed atomistic description of the NTs interaction landscape is of urgent need, aiming at the identification of novel molecules as potential therapeutics and considering the wide range of potential pharmacological applications for NGF and its family members. This mini-review will focus on the unique cartography casting the interactions of the endogenous ligand ATP, in the interaction with NGF as well as with its precursor proNGF. These interactions revealed interesting features of the ATP binding and distinct differences in the binding mode between the highly structured mature NGF and its precursor, proNGF, which is characterized by an intrinsically unstructured domain. The overview on the recent available data will be presented, together with the future perspectives on the field. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small‐molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro‐peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+, known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X‐ray scattering and hydrogen‐deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro‐peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis. [ABSTRACT FROM AUTHOR]

Patients with hematological malignancies and past serological evidence of hepatitis B are at risk for HBV reactivation. In myeloproliferative neoplasms, continuous treatment with the JAK 1/2 inhibitor ruxolitinib confers a moderate risk of reactivation (1-10%); nevertheless, no prospective randomized data are available to strongly recommend HBV prophylaxis in these patients. Here, we report a case of primary myelofibrosis and past serological evidence of HBV infection, treated with ruxolitinib and concomitant lamivudine, developing HBV reactivation due to premature withdrawal of prophylaxis. This case underlines the potential need for persistent HBV prophylaxis in the setting of ruxolitinib treatment. [ABSTRACT FROM AUTHOR]

The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF aD11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions toward the solution of this problem. [ABSTRACT FROM AUTHOR]

Background: Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A “painless” human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the “painless” hNGF mutants has not been quantified. Objectives and Results: Aiming at the therapeutic application of the “painless” hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency. Conclusions: This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF “painless” mutants. [ABSTRACT FROM AUTHOR]

Background Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of wellknown NGF-regulated mRNAs. Results To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. Conclusions Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands. [ABSTRACT FROM AUTHOR]

Background: The Raltegravir Switch for Toxicity or Adverse Events (RASTA) Study is a 2-arm randomized pilot study exploring the safety and efficacy at 48 weeks of a treatment switch to raltegravir associated with tenofovir/emtricitabine or abacavir/lamivudine in patients with regimens with optimal virological control. Methods: Patients treated with stable protease inhibitor (PI)-, non-nucleoside reverse transcriptase inhibitor (NNRTI)-, or nucleoside reverse transcriptase inhibitor (NRTI)-based regimens, with HIV-RNA levels < 50 copies/ml for ≥ 3 months and a CD4 cell count > 200 cells/μl were eligible. Enrollment of 40 patients was planned: at baseline patients were randomized 1:1 to switch to raltegravir plus tenofovir/emtricitabine (arm A) or abacavir/lamivudine (arm B). Laboratory parameters, raltegravir plasma levels, self- reported adherence, quality of life parameters, neurocognitive performance, bone composition, and body fat distribution were monitored. Virological failure was defined as HIV-RNA > 50 copies/ml on 2 consecutive determinations. Results: After 48 weeks, 5/40 (12.5%) regimen discontinuations occurred: 2 were for low-level viremia virological failure (both in arm A, at weeks 24 and 48) and 3 were for adverse events (neurological disturbances and skin rash in arm B; proximal tubulopathy in arm A). Overall, a significant CD4 increase was observed at weeks 36 and 48, and a significant decrease in total cholesterol, non-high density lipoprotein cholesterol, and triglycerides was observed at each study visit. Physical health/satisfaction in therapy scores and neuropsychological performance improved. The lumbar column Z-score improved, with no modification in other bone composition and fat distribution parameters. Conclusions: The investigated switch strategy was associated with rare virological failure. Improvements in lipid levels, quality of life measures, neuropsychological performance, and bone composition suggest good tolerability of raltegravir-based regimens. [ABSTRACT FROM AUTHOR]

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and antiamyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including b-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the bactin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs. [ABSTRACT FROM AUTHOR]

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor. [ABSTRACT FROM AUTHOR]

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a "less potent NGF" is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a "pure proNGF" signalling, distinct from the "pure NGF" one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs. [ABSTRACT FROM AUTHOR]

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X-ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75NTR receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular ' crab-like' and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the ' crab-like' to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75NTR receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners. Proteins 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In the past thirty years, nerve growth factor (NGF) has received much attention for its potential role as a therapeutic agent for Alzheimer's disease (AD) due to its neurotrophic activities on basal forebrain cholinergic neurons. This attention has been renewed by recent findings that provide new causal links between defects in NGF signaling, transport or processing to the activation of the amyloidogenic route and, more generally, to AD neurodegeneration. Thus, the concept of therapeutic administration of human recombinant NGF in AD patients has a strong rationale, being further validated by recent and ongoing clinical trials with a gene-therapy approach. However, the widespread clinical application of gene or cell-therapy strategies for the delivery of NGF to AD patients seems unpractical, and it would be more advantageous to have non-invasive methods, that should also limit the adverse effects of NGF in activating nociceptive responses. This review will describe: 1) the data from preclinical and clinical studies underlying the rationale of NGF as a potential therapeutic agent for AD; and 2) the alternative strategies to reach adequate concentrations of NGF in relevant brain areas while preventing the onset of adverse effects. [ABSTRACT FROM AUTHOR]

Discusses the role of oxyanions forming elements in waste combustion to antimony. Indications from experimental investigations regarding the presence of antimony; Factor responsible for the presence of antimony.

Experiments with cell cultures and animal models have provided solid support for the assumption that Nerve Growth Factor (NGF) plays a key role in the regulation of neuronal cell survival and death. Recently, endogenous ligands have been proposed as physiological modulators of NGF biological activity as part of this regulatory cascade. However, the structural and mechanistic determinants for NGF bioactivity remain to be elucidated. We recently unveiled, by an integrated structural biology approach, the ATP binding sites of NGF and investigated the effects on TrkA and p75NTR receptors binding. These results pinpoint ATP as a genuine endogenous modulator of NGF signaling, paving the way to the characterization of not-yet-identified chemical diverse endogenous biological active small molecules as novel modulators of NGF. The present review aims at providing an overview of the currently available 3D structures of NGF in complex with different small endogenous ligands, featuring the molecular footprints of the small molecules binding. This knowledge is essential for further understanding the functional role of small endogenous ligands in the modulation of neurotrophins signaling in physiological and pathological conditions and for better exploiting the therapeutic potentialities of NGF. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75 NTR , and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg 2+ , known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: Raltegravir (RAL) is considered one of the better-tolerated antiretroviral medications, due to limited side effects and minimal drug-drug interactions. Matherials and Methods: We retrospectively evaluated 96 HIV+, over 60 years old, experienced patients who had switched from any antiretroviral drug to raltegravir-based nuc-sparing or standard nucleoside-backbone regimens. A control group with patients aged under 60 years old was included., Results: The median age of the patients was 66 years (IQR 10.5) (77 M, 19 F); the median time horizon of follow-up was 4 years (IQR 5). HIV-RNA at baseline was undetectable for more than 6 months in most of the patients. Median CD4+ count was 453 cells/mmc (IQR 379). 49 patients had AIDS history. All the patients were assuming concomitant medications. No adverse effect attributed to the use of raltegravir was reported in the medical records. Only 2 patients presented virological failure, whereas viremic blips were observed in 10 patients. After switching to RAL-containing regimens triglycerides values showed a statistically significant reduction from a median value of 172 (IQR 105.5) mg/dl to 129 mg/dl (IQR 73) (p=0,0001). Switching to a standard regimens was associated with a marked reduction of triglycerides. Cholesterol levels were reduced at the time of follow-up (T2) but no significant modifications were observed when patients which had introduced drugs to treat dislypidemia were removed from the analysis; in contrast, triglycerides reduction was also confirmed in this sub-group. Patients presented higher levels of CD4+ at T2 and reduced platelet count [from 230 300/mmc (SD 123 527) to 197 125/mmc (SD 66 377), p=0,04]. Similar trends were observed in younger patients., Conclusion: RAL-containing regimens are safe and highly effective in the older population. RALtreatment is associated with the reduction of triglycerides and platelets count in the older population., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early '90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody αD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

Introduction: Among the range of therapeutic protein candidates for new generation treatments of neurological diseases, neurotrophic factors and recombinant antibodies hold the greatest potential. However, major difficulties in their safe and effective delivery to the brain severely limit these applications. The BBB restricts the exchange of proteins between the plasma and the CNS. Moreover, therapeutic proteins often need to be selectively targeted to the brain, while minimizing their biodistribution to systemic compartments, to avoid peripheral side effects. The intranasal delivery of proteins has recently emerged as a non-invasive, safe and effective method to target proteins to the CNS, bypassing the BBB and minimizing systemic exposure., Areas Covered: We critically summarize the main experimental and mechanistic facts about the simple and non-invasive nasal delivery approach, which provides a promising strategy and a potential solution for the severe unmet medical need of safely and effectively delivering protein therapeutics to the brain., Expert Opinion: The intranasal route for the effective delivery of recombinant therapeutic proteins represents an emerging and promising non-invasive strategy. Future studies will achieve a detailed understanding of pharmacokinetic and mechanisms of delivery to optimize formulations and fully exploit the nose-to-brain interface in order to deliver proteins for the treatment of neurological diseases. This expanding research area will most likely produce exciting results in the near future towards new therapeutical approaches for the CNS.

It is necessary to understand the molecular nature of the virus population that persists in cellular reservoirs. To achieve this we planned to characterize the patterns of resistance of HIV-1 in CD14(+) monocytes, CD4(+) T cells, and plasma. Blood samples were collected from 42 patients treated for HIV: 32 were in virological failure and in 10 viremia was undetectable. CD14(+) and CD4(+) T cells were isolated using magnetic beads. Genotyping of the reverse transcriptase and protease gene of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Of the 32 patients in virological failure, 24 (75%) had resistance mutations in at least one compartment. The numbers and types of mutations from monocytes were the same as those detected in both CD4(+) T cell-associated virus and plasma in only 8% whereas in 71% monocytes exhibited a different mutation pattern. In 21% of patients, the profile of drug-resistant mutations in the virus from blood monocytes was identical to that in plasma but differed from that in CD4. In the 71% of patients with virological suppression, the genotypic resistance pattern differed between monocytes and CD4(+) T cells. Circulating monocytes may harbor a viral dominant population different from those viruses circulating in blood and archived in CD4(+) T cells. Hence, monocytes and other cellular reservoirs might serve as an indirect source of a drug-resistant viral variant.