Parkinson’s disease (PD) is a multisystemic disorder that manifests through motor and non-motor symptoms. Motor dysfunction results from the degeneration of dopamine-producing neurons in the substantia nigra pars compacta. Increasing evidence suggests that synapse dysfunction precedes neuronal loss by years. Still, early synaptic alterations in PD remain poorly understood. Here, we integrate literature meta-analysis and multi-omics with biochemical, imaging, and electrophysiological measurements in Lrrk2 mouse models and human iPSC-derived neurons lacking LRRK2. We demonstrate that brain-derived neurotrophic factor (BDNF) activates LRRK2 in differentiated SH-SY5Y cells and primary mouse neurons, reshaping the LRRK2 interactome toward a network of actin cytoskeleton-related proteins. Gene-ontology analyses of both literature-curated LRRK2 interactors and phospho-proteome from striatal tissues with elevated LRRK2 activity highlight synapse-actin remodeling as major affected pathways. We further observed that loss of LRRK2 impairs BDNF signaling and alters postsynaptic density architecture. Young Lrrk2 knockout mice display structural alterations in dendritic protrusions, a phenotype that normalizes with age. In human iPSC-derived neurons, LRRK2 knockout affects maturation and BDNF-dependent regulation of spontaneous synaptic activity. Taken together, our study discloses a critical role of LRRK2 in BDNF-dependent synaptic modulation and identifies the synaptic actin cytoskeleton as a convergent site of LRRK2-associated pathophysiological processes in PD.

Intrinsically disordered proteins (IDPs) represent a family of proteins that given their structural peculiarity, that is, absence of defined secondary or tertiary structures, are very flexible and display great adaptability in binding to other molecules. For these reasons, IDPs are often key nodes in modulatory molecular networks. While 3D-structured proteins undergo aggregation and precipitation in denaturing conditions, due to the exposure of their hydrophobic core to the aqueous environment, IDPs are instead refractory to aggregation due to their naturally disordered state. This peculiarity has prompted the use of harsh conditions (i.e., heat or low pH) to selectively extract IDPs from complex protein samples. Here we provide the first comprehensive comparison and evaluation of the two strategies commonly adopted to enrich for IDPs, that is, heat- and perchloric acid-based extractions. We find that both methods allow the study of the intrinsically disordered "dark side" of the proteome, while displaying acute differences in their ability to enrich these peculiar IDPs.

Abstract Resistance to targeted therapy in HER2-positive breast cancer remains a clinical challenge, especially for patients with relapsed or metastatic disease. Particularly, persistent activation of hypoxia-inducible factor 1 (HIF-1) signalling is well documented in the context of trastuzumab and trastuzumab emtansine resistance. To achieve a deeper understanding of how HIF-1 activity modulates the response to anti-HER2 treatment, we functionally characterized a cellular model of hypoxia-induced drug resistance for HER2-positive breast cancer using shotgun proteomics. By global phosphoproteomics profiling, the Rac1 pathway was identified as one of the most enriched signalling networks under hypoxia. Furthermore, the selective Rac1 blockade with the 1A-116 small-molecule inhibitor sensitised HER2-positive cells to trastuzumab in both 2D and 3D culture systems. Altogether, our findings demonstrate that hypoxic conditions induce the resistance of HER2-positive breast cancer cells to targeted therapy and suggest the therapeutic potential of Rac1 inhibition to enhance trastuzumab efficacy. Highlights Hypoxic conditions induce trastuzumab resistance in HER2-positive breast cancer. Rac1 signalling was mapped under hypoxia by phosphoproteomics profiling. Rac1 inhibition sensitises HER2-positive cells to trastuzumab.

The increasing global population and evolving dietary preferences demand sustainable and innovative food solutions. Edible filamentous fungi offer a promising protein source and are emerging as key ingredients in meat alternatives. This study investigates the quality and safety of Neurospora intermedia biomass (NIB), produced in a demo-scale bubble column reactor using grape marc (GM) and wine lees (WL) as cultivation media, and synthetic glucose medium (SYN) as a control. For submerged fermentation, GM and WL were used at 4% w/v and 50% v/v, respectively, whereas 1.5% w/v glucose was used for SYN. The NIB exhibited a complete amino acid profile, with notable levels of lysine (9.25-9.46%) and leucine (8.69-9.04%), and its lipid fraction was rich in unsaturated fatty acids-oleic (41.0-59.8%) and linoleic (19.7-33.9%), along with phosphorus (1097-2747 mg/100 g) and polyphenols (5.48-7.89 mg GAE/g). Overall, the proteomic analysis allowed the identification of more than 3000 proteins. Bioinformatic predictions identified potential allergens, which together accounted for only a minor fraction of the total protein mass. This study underscores NIBs grown on GM and WL as a functional and nutritious ingredient that has a rich and diverse protein profile with potentially low allergenicity. Additionally, it offers an innovative strategy that adds economic value to oenological by-products, reduces environmental impact, and promotes a circular bioeconomy.

Bevacizumab was the first available anti-angiogenic therapy with a monoclonal antibody, initially approved against colorectal cancer. It is now used intravenously in the treatment of several other malignancies and in combination with chemotherapy. It binds to all circulating, soluble VEGF-A isoforms, inhibiting angiogenesis, and thereby reducing tumor vascularization. During storage, transport, dilution and administration, Bevacizumab is exposed to different stressors, including ambient light, which can compromise its efficacy and safety. Here, the chemico-physical instability induced by real-life doses of light on Bevacizumab-bvzr is reported in relation to the observed target recognition, anti-angiogenic activity and in vitro immunogenicity. By irradiating the diluted formulation with real-life light doses, mimicking the IV bag infusions, no conformational changes were found by UV and CD spectroscopy. However, light-exposed Bevacizumab-bvzr exhibited a marked reduction in VEGF-A binding, along with decreased anti-angiogenic efficacy in HUVEC-based assays. This loss of activity can be explained by the formation of a small amount of high molecular weight aggregates, detected by SEC, SDS-PAGE and TEM analyses, in all irradiated samples and in a concentration-dependent manner. The immunogenic properties of Bevacizumab aggregates were assessed on human monocytes-derived dendritic cells, which revealed no evidence of dendritic cell activation in vitro. At very high light doses, highlighting the amino acid modifications, mono/dioxidations and deamidation were detected by LC-MS fingerprinting analysis, involving Trp and Met, and Asn and Gln, respectively. Serine formylation and tyrosine oxidation mostly in 5 different peptides were also found. Notably, none of the identified modifications appeared within the complementarity-determining regions (CDRs). Although Bevacizumab is typically exposed to low levels of artificial and indoor light under clinical conditions, our findings indicate that it should be always protected from light. This is especially true during long IV administration periods, to avoid aggregate formation with potential reduction of the mAb therapeutic activity.

Parkinson’s disease (PD) is a multisystemic disorder that manifests through motor and non-motor symptoms. Motor dysfunction is the most debilitating and it is caused by the degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc). A body of evidence indicates that synapse demise precedes by years neuronal death. Still, early synaptic dysfunctions in PD are poorly deciphered. Here we combined literature metanalysis, proteomics and phosphoproteomics with biochemical, imaging and electrophysiological measurements in neurons, brains and synaptosomes from knockout and knockin mouse models, as well as human iPSC-derived neurons associated with the PD-kinase LRRK2. We show that phosphorylation of LRRK2 at Ser935, which controls LRRK2 subcellular localization, rapidly increases upon brain-derived neurotrophic factor (BDNF) stimulation of differentiated SH-SY5Y cells and primary mouse neurons. Affinity-purification coupled with mass spectrometry (AP-MS/MS) analysis revealed that LRRK2 interactome is significantly reshaped upon BDNF stimulation, with an interconnected network of actin cytoskeleton-associated proteins increasing their binding to LRRK2. Accordingly, LRRK2 knockout neurons exhibit decreased TrkB signaling and fail to induce BDNF-dependent spinogenesis. In vivo , one-month old Lrrk2 knockout mice display defects in spine maturation, a phenotype that disappears with age. In human iPSC-derived cortical neurons, BDNF increases the frequency of miniature excitatory post-synaptic currents (mEPSC) in wild-type but not in the presence of LRRK2 knockout, functionally supporting a distinctive role of LRRK2 in BDNF-synaptic signaling. Finally, Lrrk2 G2019S PD mutant synaptosomes display differentially phosphorylated proteins enriched in categories related to postsynaptic structural organization. Taken together, our study discloses a critical function of LRRK2 in BDNF-dependent synaptic processes and identifies the postsynaptic actin cytoskeleton as a convergent site of LRRK2 pathophysiological activity.

Micro and nanoplastics (NPs) might enter in human body and with proteins resulting in the formation of the protein corona. In this study, we investigated the formation of protein coronas on negatively and positively charged polystyrene (PS) NPs and examined their effects on the sorption of Pb²⁺ and Cd²⁺. Both positively and negatively charged PS-NPs exhibited nearly identical protein corona compositions, irrespective of surface charge, except for glycoproteins, which showed significantly higher adsorption on negatively charged particles. In ultrapure water without a protein corona, no significant adsorption of Cd²⁺ or Pb²⁺ was observed on either particle type. In human serum over 72 h, positively charged PS-NPs reduced Pb²⁺ concentration in the surrounding medium from 100 ± 4.42 % to 85.9 ± 7.63 % and Cd²⁺ from 100 ± 5.88 % to 88 ± 6.59 %, indicating sorption of these metals onto corona-coated particles. Negatively charged PS-NPs decreased Pb²⁺ to 83 ± 3.09 %. These results demonstrate that protein corona formation markedly modifies the metal sorption capacity of PS-NPs. Furthermore, Pb²⁺ consistently exhibited stronger adsorption than Cd²⁺, suggesting a metal-specific affinity of PS-NPs following corona formation. These findings demonstrated that the surface charge does not play a major role in the formation of the protein corona on nanoplastics. However, the protein corona can significantly promote the heavy metals adsorption by forming stable nanoplastics-protein corona (NP-PC) metal complexes under physiological conditions. This interaction may increase nanoplastics toxicity by enhancing the accumulation and transport of highly toxic metals within biological systems, with important implications for risk assessment.

14-3-3 constitute a highly conserved family of proteins that participate in the regulation of essential cellular processes by establishing extensive protein-protein interactions. Consequently, perturbation of the 14-3-3s interactome can be implicated in the pathogenesis of several diseases. Phosphorylation has emerged as a key mechanism by which 14-3-3s interactome can be regulated, and aberrantly phosphorylated 14-3-3s have been observed in patients with neurological disorders, among which Parkinson’s disease. Here, we specifically investigate phosphorylation of 14-3-3γ at Ser59 and assess its impact on α-Synuclein aggregation. Consistent with observations performed by others with different 14-3-3 isoforms, we observed that phosphorylation reduces the protective abilities of 14-3-3γ both in vitro and in cellular models. Importantly, this effect is also evident in cerebrospinal fluid (CSF), suggesting that phosphorylation of 14-3-3γ may directly contribute to α-Synuclein aggregation in vivo and may be relevant to disease-associated mechanisms. Using bioinformatics approaches, we further examined how Ser59 phosphorylation reshapes the global 14-3-3γ interactome, revealing novel interaction networks and pathways potentially implicated in neurodegenerative disease pathogenesis.

Summary Coding mutations in the Leucine-rich repeat kinase 2 ( LRRK2 ) gene represent the most common cause of familial Parkinson’s disease (PD), and are frequently observed in idiopathic PD. In addition, variation around the LRRK2 locus has been shown to alter PD risk by genome-wide association studies. Disease-causing mutations cluster within the catalytic core of LRRK2 – composed of GTPase (ROC) and serine-threonine kinase domains – and lead to an increase in kinase activity, resulting in hyperphosphorylation of a subset of RAB GTPases and consequent cellular toxicity. However, the interplay between LRRK2 GTPase and kinase domains, and with the surrounding scaffold regions has remained underexplored, with implications for the prediction of on- and off-target effects associated with kinase inhibition. To address this gap, here we dissected the contributions of kinase, GTPase and scaffold domains to LRRK2 function in murine macrophages and tissues expressing endogenous levels of GTP/GDP-binding deficient Lrrk2 T1348N. Guanosine nucleotide-free Lrrk2 is devoid of both GTPase and kinase activities but maintains the scaffold shell, leading to significant reshaping of Lrrk2 interactome and engagement in novel interactions. This altered functional state leads to impaired autophagy and accumulation of enlarged lysosomes and autophagic cargo in macrophages and kidneys. Since pharmacological inhibition of LRRK2 is under clinical evaluation, our results reveal retained scaffold functions upon loss of catalytic activity that warrant careful consideration.

Mastitis is the most important bovine disease, causing dramatic economic losses to the dairy industry, worldwide. This study explores the valorization of whey from cows affected by mastitis, through a novel separation approach. Surface Active Maghemite Nanoparticles (SAMNs) were used as magnetic baits to selectively bind bioactive peptides with potential health benefits. Advanced techniques such as HPLC and LC-MS/MS highlighted SAMN capability of isolating a restricted group of peptides, drastically diverging from the control profile (Solid Phase Extraction, SPE) and characterized by a peculiar acidic residue distribution. Most importantly, both magnetically purified and nano-immobilized peptides (SAMN@peptides) showed protective activity against oxidative stress and inflammation, when tested on Caco-2 cells; with SAMN@peptides being associated with the strongest biological effect. SAMNs exhibited excellent characteristics, they are environmentally sustainable, and their synthesis is cost-effective prompting at a scalable and selective tool for capturing bioactive peptides, with potential applications in functional foods and nutraceuticals.

Abstract Parkinson’s disease (PD) is a multisystemic disorder that manifests through motor and non-motor symptoms. Motor dysfunction is the most debilitating and it is caused by the degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc). Increasing evidence suggests that synapse dysfunction precedes neuronal loss by years. Still, early synaptic alterations in PD remain poorly understood. Here we integrate literature meta-analysis, proteomics and phosphoproteomics with biochemical, imaging and electrophysiological measurements in neurons and brains from knockout and knockin Lrrk2 mouse models, as well as human iPSC-derived neurons lacking LRRK2. We demonstrate that phosphorylation of LRRK2 at Ser935 and of RAB proteins is induced by brain-derived neurotrophic factor (BDNF) stimulation in differentiated SH-SY5Y cells and primary mouse neurons. Affinity-purification coupled with mass spectrometry (AP-MS/MS) revealed a significant remodelling of the LRRK2 interactome following BDNF treatment, with enhanced association of LRRK2 to a network of actin cytoskeleton-related proteins. Gene-ontology analyses of both literature-curated LRRK2 interactors and phospho-proteome from striatal tissues with elevated LRRK2 activity (G2019S knockin mice) highlight synapse-actin remodelling as major affected pathways. We further observed that loss of LRRK2 impairs BDNF signaling and alters postsynaptic density architecture. One month-old Lrrk2 knockout mice display structural alterations in dendritic protrusions, a phenotype that normalizes with age. In human iPSC-derived neurons, BDNF enhances the frequency of miniature excitatory post-synaptic currents (mEPSC) in wild-type but not in LRRK2 knockout neurons, which appear to bypass this regulation through developmental compensation. Taken together, our study discloses a critical role of LRRK2 in BDNF-dependent synaptic modulation and identifies the synaptic actin cytoskeleton as a convergent site of LRRK2’s pathophysiological activity.

Inflammatory bowels diseases (IBD) are high risk conditions for colorectal cancer (CRC). The discovery of IBD and CRC noninvasive protein/peptide biomarkers using saliva and feces was the aim of this study involving 20 controls, 25 IBD (12 Crohn's Disease-CD), 37 CRC. By untargeted proteomic (LTQ-Orbitrap/MS), a total of 152 proteins were identified in saliva. Absent in controls, 73 proteins were present in both IBD and CRC, being mainly related to cell-adhesion, cadherin-binding and enzyme activity regulation (g-Profiler). Among the remaining 79 proteins, 14 were highly expressed in CD and 11 in CRC. These proteins clustered in DNA replication/expression and innate/adaptive immunity. In stool, endogenous peptides from 30 different proteins were identified, two being salivary and CD-associated: Basic Proline-rich Protein 1 (PRBs) and Acidic Proline-rich Phosphoprotein. Biological effects of the PRBs-related peptides GQ-15 and GG-17 found in CD stool were evaluated using CRC cell lines. These peptides induced cell proliferation and activated Erk1/2, Akt and p38 pathways. In conclusion, the salivary proteome unveiled DNA stability and immunity clusters shared between IBD and CRC. Salivary PRB-derived peptides, enriched in CD stool, stimulate CRC cell proliferation and the pro-oncogenic RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways suggesting a potential involvement of PRBs in IBD and cancer pathogenesis.

Porphyrins belong to a peculiar class of natural molecules involved in many different biological processes of paramount importance, from photosynthesis in green plants, to the oxygen binding and transportation in breathing and to the oxidation reactions catalyzed by the enzymatic family of cytochromes P450. This versatility is due to the tight structure/properties relationship of these macrocycles. Many different synthetic strategies have been used to modify the porphyrin core by connecting electro- and photo-active moieties in both meso- and beta-pyrrole positions. Among them all, C-C bond formation still remains one of the most useful approaches to ensure fine control over the chemical-physics properties of porphyrins. In this work we successfully tested the catalytic system Pd(dba)(2)/AsPh(3 )in the Heck cross-coupling reaction between beta-monobrominated tetraphenylporphyrin and ferrocenyl-alkene derivatives, avoiding the use of excess alkene and Ni(ii) or Zn(ii) porphyrinate complexes. The preferential formation of the alpha- versus trans-isomer was observed when vinylferrocene was used as the starting alkene, compared to styrene and 4-ferrocenylstyrene, either at lower (60 degrees C) or higher (90 degrees C) reaction temperature. Subsequent electrochemical and photochemical studies on ferrocene-porphyrin dyads pointed out the importance of the type of beta-C-C bond between the two chromophores in modulating/affecting the electron-transfer processes. Such studies can be useful for the realization of highly sensible fluorescent-redox switches or even in the development of highly efficient ferrocene/porphyrin-based chemodynamic therapy agents.

Cyclic poly(2-methyl-2-oxazine) (c-PMOZI) brush shells on Au nanoparticles (NPs) exhibit enhanced stealth properties toward serum and different cell lines compared to their linear PMOZI (l-PMOZI) counterparts. While selectively recruiting immunoglobulins, c-PMOZI shells reduce overall human serum (HS) protein binding and alter the processing of complement factor 3 (C3) compared to chemically identical linear shells. Polymer cyclization significantly decreases NP uptake by nonphagocytic cells and macrophages in both complement-deficient fetal bovine serum (FBS) and complement-expressing HS, indicating ineffective functional opsonization. Even in serum-free media, c-PMOZI-coated NPs show reduced internalization by macrophages compared to l-PMOZI-coated NPs, suggesting lower opsonin-independent cell surface affinity. This study demonstrates that cyclic PMOZI suppresses interactions of NPs with proteins and cells, highlighting how control over chain topology expands the polymer chemistry toolbox for modulating the behavior of core-shell NPs within physiological environments.

The evolving field of food technology is increasingly dedicated to developing functional foods. This study explored bioactive peptides from sunflower protein isolate (SPI), obtained from defatted flour, a by-product of the oil processing industry. SPI underwent simulated gastrointestinal digestion and the obtained peptide-enriched fraction (PEF) showed antioxidant properties in vivo, in zebrafish. Among the peptides present in PEF identified by mass spectrometry analysis, we selected those with antioxidant properties by in silico evaluation, considering their capability to interact with Keap1, key protein in the regulation of antioxidant response. The selected peptides were synthesized and evaluated in a cellular model. As a result, DVAMPVPK, VETGVIKPG, TTHTNPPPEAE, LTHPQHQQQGPSTG and PADVTPEEKPEV activated Keap1/Nrf2 pathway leading to Antioxidant Response Element-regulated enzymes upregulation. Since the crosstalk between Nrf2 and NF-κB is well known, the potential anti-inflammatory activity of the peptides was assessed and principally PADVTPEEKPEV showed good features both as antioxidant and anti-inflammatory molecule.

Life cycle of the dimorphic sugarcane smut fungi, Sporisorium scitamineum , involves recognition and mating of compatible saprophytic yeast -like haploid sporidia (MAT -1 and MAT -2) that upon fusion, develop into infective dikaryotic mycelia. Although the dimorphic transition is intrinsically linked with the pathogenicity and virulence of S. scitamineum , it has never been studied using a proteomic approach. In the present study, an iTRAQ-based comparative proteomic analysis of three distinct stages was carried out. The stages were: the dimorphic transition period - haploid sporidial stage (MAT -1 and MAT -2); the transition phase (24 h post co -culturing (hpc)) and the dikaryotic mycelial stage (48 hpc). Functional categorization of differentially abundant proteins showed that the most altered biological processes were energy production, primary metabolism, especially, carbohydrate, amino acid, fatty acid, followed by translation, post -translation and protein turnover. Several differentially abundant proteins (DAPs), especially in the dikaryotic mycelial stage were predicted as effectors. Taken together, key molecular mechanisms underpinning the dimorphic transition in S. scitamineum at the proteome level were highlighted. The catalogue of stage -specific and dimorphic transition -associated -proteins and potential effectors identified herein represents a list of potential candidates for defective mutant screening to elucidate their functional role in the dimorphic transition and pathogenicity in S. scitamineum . Biological significance: Being the first comparative proteomics analysis of S. scitamineum , this study comprehensively examined three pivotal life cycle stages of the pathogen: the non-pathogenic haploid phase, the transition phase, and the pathogenic dikaryotic mycelial stage. While previous studies have reported the sugarcane and S. scitamineum interactions, this study endeavored to specifically identify the proteins responsible for pathogenicity. By analyzing the proteomic alterations between the haploid and dikaryotic mycelial phases, the study revealed significant changes in metabolic pathway -associated proteins linked to energy production, notably oxidative phosphorylation, and the citrate cycle. Furthermore, this study successfully identified key metabolic pathways that undergo reprogramming during the transition from the non-pathogenic to the pathogenic stage. The study also deciphered the underlying mechanisms driving the morphological and physiological alterations crucial for the S. scitamineum virulence. By studying its life cycle stages, identifying the key metabolic pathways and stage -specific proteins, it provides unprecedented insights into the pathogenicity and potential avenues for intervention. As proteomics continues to advance, such studies pave the way for a deeper understanding of plantpathogen interactions and the development of innovative strategies to mitigate the impact of devastating pathogens like S. scitamineum.