RESUMO
BACKGROUND: Biosurfactants are surface-active compounds with environmental and industrial applications. These molecules show higher biocompatibility, stability and efficiency compared to synthetic surfactants. On the other hand, biosurfactants are not cost-competitive to their chemical counterparts. Cost effective technology such as the use of low-cost substrates is a promising approach aimed at reducing the production cost. This study aimed to evaluate the biosurfactant production and activity by the novel strain Rhodococcus sp. SP1d by using different growth substrates. Therefore, to exploit the biosurfactant synthesized by SP1d for environmental applications, the effect of this compound on the bacteria biofilm formation was evaluated. Eventually, for a possible bioremediation application, the biosurfactant properties and its chemical characteristics were investigated using diesel as source of carbon. RESULTS: Rhodococcus sp. SP1d evidence the highest similarity to Rhodococcus globerulus DSM 43954T and the ability to biosynthesize surfactants using a wide range of substrates such as exhausted vegetable oil, mineral oil, butter, n-hexadecane, and diesel. The maximum production of crude biosurfactant after 10 days of incubation was reached on n-hexadecane and diesel with a final yield of 2.38 ± 0.51 and 1.86 ± 0.31 g L- 1 respectively. Biosurfactants produced by SP1d enhanced the biofilm production of P. protegens MP12. Moreover, the results showed the ability of SP1d to produce biosurfactants on diesel even when grown at 10 and 18 °C. The biosurfactant activity was maintained over a wide range of NaCl concentration, pH, and temperature. A concentration of 1000 mg L- 1 of the crude biosurfactant showed an emulsification activity of 55% towards both xylene and olive oil and a reduction of 25.0 mN m- 1 of surface tension of water. Eventually, nuclear magnetic resonance spectroscopy indicated that the biosurfactant is formed by trehalolipids. CONCLUSIONS: The use of low-cost substrates such as exhausted oils and waste butter reduce both the costs of biosurfactant synthesis and the environmental pollution due to the inappropriate disposal of these residues. High production yields, stability and emulsification properties using diesel and n-hexadecane as substrates, make the biosurfactant produced by SP1d a sustainable biocompound for bioremediation purpose. Eventually, the purified biosurfactant improved the biofilm formation of the fungal antagonistic strain P. protegens MP12, and thus seem to be exploitable to increase the adherence and colonization of plant surfaces by this antagonistic strain and possibly enhance antifungal activity.
Assuntos
Alcanos , Rhodococcus , Tensoativos/química , Tensão Superficial , Biodegradação AmbientalRESUMO
Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography-electrospray ionization-tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH.
Assuntos
Peptídeos , Hidrolisados de Proteína , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Colágeno/química , Peptídeos/química , Hidrolisados de Proteína/químicaRESUMO
Two years since the outbreak of the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS-CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side effect. One alternative, with structural similarities to heparin, is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated, and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective in inhibiting cell infection than low-molecular-weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.
Assuntos
Poliéster Sulfúrico de Pentosana , SARS-CoV-2 , Ligação Viral , Animais , Anticoagulantes/farmacologia , Chlorocebus aethiops , Heparina/uso terapêutico , Poliéster Sulfúrico de Pentosana/farmacologia , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus , Células Vero , Ligação Viral/efeitos dos fármacosRESUMO
The persistence of neurological symptoms after SARS-CoV-2 infection, as well as the presence of late axonal damage, is still unknown. We performed extensive systemic and neurological follow-up evaluations in 107 out of 193 consecutive patients admitted to the COVID-19 medical unit, University Hospital of Verona, Italy between March and June 2020. We analysed serum neurofilament light chain (NfL) levels in all cases including a subgroup (n = 29) of patients with available onset samples. Comparisons between clinical and biomarker data were then performed. Neurological symptoms were still present in a significant number (n = 49) of patients over the follow-up. The most common reported symptoms were hyposmia (n = 11), fatigue (n = 28), myalgia (n = 14), and impaired memory (n = 11) and were more common in cases with severe acute COVID-19. Follow-up serum NfL values (15.2 pg/mL, range 2.4-62.4) were within normal range in all except 5 patients and did not differentiate patients with vs without persistent neurological symptoms. In patients with available onset and follow-up samples, a significant (p < 0.001) decrease of NfL levels was observed and was more evident in patients with a severe acute disease. Despite the common persistence of neurological symptoms, COVID-19 survivors do not show active axonal damage, which seems a peculiar feature of acute SARS-CoV-2 infection.
Assuntos
Axônios/patologia , COVID-19/patologia , Doenças do Sistema Nervoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ageusia/patologia , Ageusia/virologia , Anosmia/patologia , Anosmia/virologia , Axônios/virologia , Progressão da Doença , Fadiga/patologia , Fadiga/virologia , Feminino , Humanos , Itália , Masculino , Transtornos da Memória/patologia , Transtornos da Memória/virologia , Pessoa de Meia-Idade , Mialgia/patologia , Mialgia/virologia , Doenças do Sistema Nervoso/virologia , Proteínas de Neurofilamentos/sangue , SARS-CoV-2RESUMO
SARS-CoV-2 survivors may report persistent symptoms that resemble myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We explored (a) ME/CFS-like symptom prevalence and (b) whether axonal, inflammatory, and/or lung changes may contribute to ME/CFS-like symptoms in SARS-CoV-2 survivors through clinical, neuropsychiatric, neuropsychological, lung function assessment, and serum neurofilament light chain, an axonal damage biomarker. ME/CFS-like features were found in 27% of our sample. ME/CFS-like group showed worse sleep quality, fatigue, pain, depressive symptoms, subjective cognitive complaints, Borg baseline dyspnea of the 6-min walking test vs. those without ME/CFS-like symptoms. These preliminary findings raise concern on a possible future ME/CFS-like pandemic in SARS-CoV-2 survivors.
Assuntos
COVID-19/complicações , Síndrome de Fadiga Crônica/epidemiologia , Síndrome de Fadiga Crônica/virologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , SARS-CoV-2RESUMO
The in vitro models are receiving growing attention in studies on skin permeation, penetration, and irritancy, especially for the preclinical development of new transcutaneous drugs. However, synthetic membranes or cell cultures are unable to effectively mimic the permeability and absorption features of the cutaneous barrier. The use of explanted skin samples maintained in a fluid dynamic environment would make it possible for an in vitro experimentation closer to in vivo physiological conditions. To this aim, in the present study, we have modified a bioreactor designed for cell culture to host explanted skin samples. The preservation of the skin was evaluated by combining light, transmission, and scanning electron microscopy, for the histo/cytological characterization, with nuclear magnetic resonance spectroscopy, for the identification in the culture medium of metabolites indicative of the functional state of the explants. Our morphological and metabolomics results demonstrated that fluid dynamic conditions ameliorate significantly the structural and functional preservation of skin explants in comparison with conventional culture conditions. Our in vitro system is, therefore, reliable to test novel therapeutic agents intended for transdermal administration in skin samples from biopsies or surgical materials, providing predictive information suitable for focused in vivo research and reducing animal experimentation.
Assuntos
Hidrodinâmica , Metabolômica , Pele , Administração Cutânea , Animais , Microscopia Eletrônica de Varredura , PermeabilidadeRESUMO
In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.
Assuntos
Cálcio/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Magnésio/metabolismo , Mutação , Distrofias Retinianas/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Humanos , Conformação Proteica , Multimerização ProteicaRESUMO
The unpredictable course and uncertain impact of relapses make treatment strategies of anti-myelin oligodendrocyte glycoprotein antibodies associated disorders (MOGAD) challenging. We analysed neurofilament light chain levels (NfL) in onset and follow-up sera of 18 patients with MOGAD to clarify the timing of axonal damage. In comparison with disease onset values (median 8.9 pg/mL, range 1.8-97), NfL levels remained stable or decreased in most follow-up measurements (n=52, median 6.7 pg/mL, range 0.2-207), including those measured on relapses. The predominant axonal damage occurs during onset, which could be the main driving factor of final disability, with subsequent relevant clinical and therapeutic implications.
Assuntos
Autoanticorpos , Humanos , Glicoproteína Mielina-OligodendrócitoRESUMO
BACKGROUND AND PURPOSE: The diagnosis of spontaneous spinal cord infarction (SCI) is limited by the lack of diagnostic biomarkers and MRI features that often overlap with those of other myelopathies, especially acute myelitis. We investigated whether the ratio between serum neurofilament light chain levels and MRI T2-lesion area (neurofilament light chain/area ratio-NAR) differentiates SCI from acute myelitis of similar severity. METHODS: We retrospectively identified Mayo Clinic patients (January 1, 2000-December 31, 2019) with (1) SCI, (2) AQP4 (aquaporin 4)-IgG or MOG (myelin oligodendrocyte glycoprotein)-IgG-associated myelitis at disease clinical presentation, or (3) idiopathic transverse myelitis from a previously identified population-based cohort of patients seronegative for AQP4-IgG and MOG-IgG. Serum neurofilament light chain levels (pg/mL) were assessed at the Verona University (SIMOA, Quanterix) in a blinded fashion on available stored samples obtained ≤3 months from myelopathy presentation. For each patient, the largest spinal cord lesion area (mm2) was manually outlined by 2 independent raters on sagittal T2-weighted MRI images, and the mean value was used to determine NAR (pg/[mL·mm2]). RESULTS: Forty-eight patients were included SCI, 20 (definite, 11; probable, 6; possible, 3); acute myelitis, 28 (AQP4-IgG-associated, 17; MOG-IgG-associated, 5; idiopathic transverse myelitis, 6). The median expanded disability status scale score (range) at myelopathy nadir were 7.75 (2-8.5) and 5.5 (2-8), respectively. Serum neurofilament light chain levels (median [range] pg/mL) in patients with SCI (188 [14.3-2793.4]) were significantly higher compared with patients with AQP4-IgG-associated myelitis (37 [0.8-6942.9]), MOG-IgG-associated myelitis (45.8 [4-283.8]), and idiopathic transverse myelitis (15.6 [0.9-217.8]); P=0.01. NAR showed the highest accuracy for identification of SCI versus acute myelitis with values ≥0.35 pg/(mL·mm2) yielding 86% specificity and 95% sensitivity (area under the curve=0.93). The positive and negative likelihood ratios were 6.67 and 0.06, respectively. NAR remained independently associated with SCI after adjusting for age, gender, immunotherapy before sampling, and days from myelopathy symptoms onset to sampling (P=0.0007). CONCLUSIONS: NAR is a novel and promising clinical biomarker for differentiation of SCI from acute myelitis.
Assuntos
Infarto/sangue , Infarto/diagnóstico por imagem , Mielite Transversa/sangue , Mielite Transversa/diagnóstico por imagem , Proteínas de Neurofilamentos/sangue , Isquemia do Cordão Espinal/diagnóstico por imagem , Isquemia do Cordão Espinal/diagnóstico , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Aquaporina 4/sangue , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Imunoterapia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Glicoproteína Mielina-Oligodendrócito/sangue , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Flowering and fruiting are processes subject to complex control by environmental and endogenous signals. Endogenous signals comprise, besides classical phytohormones, also signaling peptides and miniproteins. Tomato cystine-knot miniproteins (TCMPs), which belong to a Solanaceous-specific group of Cys-rich protein family, have been recently involved in fruit development. TCMP-1 and TCMP-2 display a highly modulated expression pattern during flower and fruit development. A previous study reported that a change in the ratio of the two TCMPs affects the timing of fruit production. In this work, to investigate TCMP-2 mode of action, we searched for its interacting partners. One of the interactors identified by a yeast two hybrid screen, was the B-box domain-containing protein 16 (SlBBX16), whose closest homolog is the Arabidopsis microProtein 1b implicated in flowering time control. We demonstrated the possibility for the two proteins to interact in vivo in tobacco epidermal cells. Arabidopsis plants ectopically overexpressing the TCMP-2 exhibited an increased level of FLOWERING LOCUS T (FT) mRNA and anticipated flowering. Similarly, in previously generated transgenic tomato plants with increased TCMP-2 expression in flower buds, we observed an augmented expression of SINGLE-FLOWER TRUSS gene, the tomato ortholog of FT, whereas the expression of the antiflorigen SELF-PRUNING was unchanged. Consistently, these transgenic plants showed alterations in the flowering pattern, with an accelerated termination of the sympodial units. Overall, our study reveals a novel function for TCMP-2 as regulatory factor that might integrate, thanks to its capacity to interact with SlBBX16, into the signaling pathways that control flowering, and converge toward florigen regulation.
RESUMO
In clinical practice, administration of low ozone (O3) dosages is a complementary therapy for many diseases, due to the capability of O3 to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O3 concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O3 treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O3 treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O3 concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O3 as an adjuvant treatment to improve fat engraftment.
Assuntos
Infecções por Coronavirus , Coronavirus , Pandemias , Pneumonia Viral , Betacoronavirus , COVID-19 , China , Humanos , Sistema Nervoso , SARS-CoV-2RESUMO
The diagnosis of peripheral neuropathies can be challenging with consequent difficulties in patients' management. The aim of this study was to explore the current diagnostic role of sural nerve biopsy and to compare pathological findings with serum neurofilament light chain levels (NfL) as biomarkers of axonal damage. We collected demographic, clinical, and paraclinical data of patients referred over 1 year to the Neurology Unit, University of Verona, Italy, to perform nerve biopsy for diagnostic purposes, and we analyzed NfL levels in available paired sera using a high sensitive technique (Quanterix, Simoa). Eighty-two patients were identified (37.8% females, median age 65.5 years). Neuropathy onset was frequently insidious (68.3%) with a slowly progressive course (76.8%). Lower limbs were usually involved (81.7%), with a predominance of sensory over motor symptoms (74.4% vs 42.7%). The most common neuropathological findings were a demyelinating pattern (76.8%), clusters of regenerations (58.5%), and unmyelinated fibers involvement on ultrastructural evaluation (52.4%). A definite pathological diagnosis was achieved in 29 cases, and in 20.7% of patients, the referral clinical diagnosis was modified. Coexistent hematological conditions and hepatitis were diagnostic confounding factors (p = 0.012 and 0.034, respectively). In the analyzed paired sera (n = 37), an inverse despite not significant relationship between NfL values and fiber density was observed (Spearman's rho - 0.312, p = 0.056). In addition, we noted increased serum NfL values of patients with active axonal degeneration. Nerve biopsy remains a useful diagnostic investigation to achieve a correct diagnosis and guide patients' management in selected cases of peripheral neuropathy. Serum NfL is an accessible and potential valuable marker of axonal damage in these conditions.
Assuntos
Filamentos Intermediários , Doenças do Sistema Nervoso Periférico , Nervo Sural , Idoso , Biópsia , Feminino , Humanos , Itália , Masculino , Proteínas de Neurofilamentos , Doenças do Sistema Nervoso Periférico/diagnóstico , Nervo Sural/patologiaRESUMO
Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased Km value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb.
Assuntos
Atrofia Girata/genética , Ornitina-Oxo-Ácido Transaminase/genética , Biocatálise , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Mutação , Ornitina-Oxo-Ácido Transaminase/químicaRESUMO
Rhodopsins are photoreceptive proteins using light to drive a plethora of biological functions such as vision, proton and ion pumping, cation and anion channeling, and gene and enzyme regulation. Here we combine organic synthesis, NMR structural studies, and photochemical characterization to show that it is possible to prepare a fully synthetic mimic of rhodopsin photoreceptors. More specifically, we conjugate a bile acid binding protein with a synthetic mimic of the rhodopsin protonated Schiff base chromophore to achieve a covalent complex featuring an unnatural protein host, photoswitch, and photoswitch-protein linkage with a reverse orientation. We show that, in spite of its molecular-level diversity, light irradiation of the prepared mimic fuels a photochromic cycle driven by sequential photochemical and thermal Z/E isomerizations reminiscent of the photocycles of microbial rhodopsins.
Assuntos
Materiais Biomiméticos/química , Proteínas de Transporte/química , Corantes/química , Glicoproteínas de Membrana/química , Fotorreceptores Microbianos/química , Rodopsina/química , Isomerismo , Luz , Processos Fotoquímicos , Conformação Proteica , Prótons , Relação Estrutura-AtividadeRESUMO
The production of Amarone wine is governed by a disciplinary guideline to preserve its typical features; however, postharvest infections by the fungus Botrytis cinerea (B. cinerea) not only represent a phytosanitary problem but also cause a significant loss of product. In this study, we tested a treatment with mild ozoniztion on grapes for Amarone wine production during withering in the fruttaio (the environment imposed by the disciplinary guideline) and evaluated the impact on berry features by a multimodal imaging approach. The results indicate that short and repeated treatments with low O3 concentrations speed up the naturally occurring berry withering, probably inducing a reorganization of the epicuticular wax layer, and inhibit the development of B. cinerea, blocking the fungus in an intermediate vegetative stage. This pilot study will pave the way to long-term research on Amarone wine obtained from O3-treated grapes.
Assuntos
Imagem Multimodal/métodos , Ozônio/farmacologia , Análise Espectral/métodos , Vitis/efeitos dos fármacos , Vinho/microbiologia , Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Botrytis/patogenicidade , Microbiologia de Alimentos , Conservação de Alimentos/métodos , Frutas/química , Itália , Imageamento por Ressonância Magnética , Microscopia Eletrônica de Varredura , Projetos Piloto , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Vitis/química , Vitis/microbiologia , Vinho/análiseRESUMO
Amyloid structures are universal structures, widely diffuse in nature. Silk, capable of forming some of the strongest tensile materials on earth represents an important example of formation of functional amyloid fibrils, a process reminiscent of the oligomerization of peptides involved in neurodegenerative diseases. The stability of silk fibroin solutions in different conditions and its transition from α-helix/random coil to ß-sheet structures, at the basis of gelation processes and fibril formation, have been here investigated and monitored employing different biophysical approaches. Silk fibroin aggregation state as a function of concentration, pH and aging has been characterized employing NMR ordered diffusion spectroscopy. The change of silk fibroin diffusion coefficient over time, which reflects the progress of oligomerization, has been monitored for silk fibroin alone and in the presence of a polycondensed aromatic dye, namely rhodamine 6G. NMR, UV and DLS measurements indicated that rhodamine specifically binds to silk fibroin with a micromolar KD. The reported data reveal, for the first time, that RHD is capable of inhibiting fibroin self-association, thus controlling ß-conformational transition at the basis of fibril formation. The described approach could be extended to further protein systems, allowing better control of the oligomerisation process.
Assuntos
Fibroínas/metabolismo , Agregados Proteicos , Rodaminas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica , Estabilidade Proteica , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
In biological systems, nanoparticles (NPs) elicit bioactivity upon interaction with proteins. As a result of post-translational modification, proteins occur in a variety of alternative covalent forms, including structural isomers, which present unique molecular surfaces. We aimed at a detailed description of the recognition of protein isomeric species by NP surfaces. The transient adsorption of isomeric ubiquitin (Ub) dimers by NPs was investigated by solution NMR spectroscopy. Lys63- and Lys48-linked Ub2 were adsorbed by large anionic NPs with different affinities, whereas the binding strength was similar in the cases of smaller particles. After the incorporation of paramagnetic tags into NPs, the observed site-resolved paramagnetic footprints provided a high-resolution map of the different protein surfaces binding to NPs. The approach described could be extended to further protein isoforms and more specialized NP systems to allow better control of the interactions between NPs and protein targets.
Assuntos
Nanopartículas/química , Proteínas/química , Ubiquitina/química , Adsorção , Isomerismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Liver fatty acid binding protein (L-FABP) is an abundant cytosolic protein playing a central role in intracellular lipid trafficking. The L-FABP T94A variant, originating from one of the most common polymorphisms in the FABP family, is associated with several lipid-related disorders. However, the molecular factors that determine the observed functional differences are currently unknown. In our work, we performed a high resolution comparative molecular analysis of L-FABP T94T and L-FABP T94A in their unbound states and in the presence of representative ligands of the fatty acid and bile acid classes. We collected residue-resolved NMR spectral fingerprints of the two variants, and compared secondary structures, backbone dynamics, side chain arrangements, binding site occupation, and intermolecular contacts. We found that threonine to alanine replacement did not result in strongly perturbed structural and dynamic features, although differences in oleic acid binding by the two variants were detected. Based on chemical shift perturbations at sites distant from position 94 and on differences in intermolecular contacts, we suggest that long-range communication networks in L-FABP propagate the effect of amino acid substitution at sites relevant for ligand binding or biomolecular recognition.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Ácido Glicocólico/metabolismo , Ácido Oleico/metabolismo , Polimorfismo de Nucleotídeo Único , Regulação Alostérica , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Ileal bile acid-binding protein, IBABP, participates in the intracellular trafficking of bile salts and influences their signaling activities. The recently discovered variant, IBABP-L, bearing an N-terminal 49-amino acid extension, was found to be associated with colorectal cancer and to protect cancer cells from the cytotoxic effects of deoxycholate. However, the precise function and the molecular properties of this variant are currently unknown. METHODS: Bioinformatics tools and confocal microscopy were used to investigate the sub-cellular localization of IBABP-L; protein dynamics, ligand binding and interaction with membrane models were studied by 2D NMR and fluorescence spectroscopy. RESULTS: Based on sub-cellular localization experiments we conclude that IBABP-L is targeted to the secretory pathway by a 24-residue signal peptide and, upon its cleavage, the mature protein is constitutively released into the extracellular space. Site-resolved NMR experiments indicated the distinct preference of primary and secondary bile salts to form either heterotypic or homotypic complexes with IBABP-L. The presence of the relatively dynamic N-terminal extension, originating only subtle conformational perturbations in the globular domain, was found to influence binding site occupation in IBABP-L as compared to IBABP. Even more pronounced differences were found in the tendency of the two variants to associate with phospholipid bilayers. CONCLUSIONS: IBABP-L exhibits different sub-cellular localization, ligand-binding properties and membrane interaction propensity compared to the canonical short isoform. GENERAL SIGNIFICANCE: Our results constitute an essential first step towards an understanding of the role of IBABP-L in bile salt trafficking and signaling under healthy and pathological conditions.