229 research outputs found
Embryo-Induced Changes in the Protein Profile of Bovine Oviductal Extracellular Vesicles
No full textThe study of early maternal-embryonic cross-talk remains one of the most challenging topics in reproductive biology. Understanding the physiological mechanisms involved in the interactions between the maternal reproductive tract and the developing embryo is essential for enhancing bovine reproductive efficiency. This complex communication starts within the oviduct, where the modulation of biological processes important for ensuring embryo quality is partially facilitated through extracellular vesicles (EVs). Utilizing a combination of in vivo and in vitro models this study had three main objectives: 1) to examine the protein cargo of EVs isolated from the oviductal fluid (OF) of cyclic and pregnant heifers to understand their role in maternal-embryonic communication in vivo; 2) to characterize the protein profile of EVs in conditioned medium (CM) resulting from the culture of oviductal explants alone (Exp) or in the presence of 8- to 16-cell stage embryos (Exp + Emb); and 3) to compare the protein cargo of EVs from Exp with EVs from cyclic heifers and EVs from Exp + Emb with EVs from pregnant heifers. Proteins were considered "identified" if detected in at least three out of five replicates and considered "exclusive" if detected in at least three out of five replicates within one group but absent in all samples of other groups. We identified 659 and 1476 proteins in the OF-EVs of cyclic and pregnant heifers, respectively. Among these, 644 proteins were identified in OF-EVs from both cyclic and pregnant heifers, and 40 proteins were exclusive to OF-EVs from the pregnant group. Within the 644 proteins identified in both groups, 31 were identified as differently abundant proteins (DAPs). In pregnant heifers, DAPs were mainly related to genome activation, DNA repair, embryonic cell differentiation, migration, and immune tolerance. In vitro, we identified 841 proteins in the CM-EVs from Exp alone, 613 from Exp + Emb, and 111 in the CM-EVs from Emb alone. In the qualitative analysis between the three in vitro groups, 81 proteins were identified in all groups, 452 were common to Exp and Exp + Emb, 17 were common to Exp and Emb, 5 were common to Exp + Emb and Emb, 4 were unique to Exp, 6 were unique to Exp + Emb, and none were unique to Emb. Proteins identified when there is an interaction between the oviduct and the embryo in vitro, corresponding to the Exp + Emb group, were associated with immune tolerance, structural activity, binding, and cytoskeletal regulation. In vivo and in vitro EVs exhibit distinct qualitative and quantitative protein contents, both when comparing EVs produced in the absence of an embryo (Cyclic and Exp) and those that have undergone embryo-oviduct interaction (Pregnant and Exp + Emb). The observed changes in the protein cargo of EVs due to maternal-embryonic communication in vivo and in vitro suggest that the interaction between the embryo and the maternal milieu initiates within the oviduct and is potentially facilitated by EVs and their protein contents.This research was supported by research projects: PID2019-111641RB-I00 and PID2023-149027OB-I00 funded by MCIN/AEI/10.13039/501100011033/ to DR and PRE2020-094452 to RM.Peer reviewe
Identification of the Wnt signal peptide that directs secretion on extracellular vesicles
No full textWnt proteins are hydrophobic glycoproteins that are nevertheless capable of long-range signaling. We found that Wnt7a is secreted long distance on the surface of extracellular vesicles (EVs) following muscle injury. We defined a signal peptide region in Wnts required for secretion on EVs, termed exosome-binding peptide (EBP). Addition of EBP to an unrelated protein directed secretion on EVs. Palmitoylation and the signal peptide were not required for Wnt7a-EV secretion. Coatomer was identified as the EV-binding protein for the EBP. Analysis of cocrystal structures, binding thermodynamics, and mutagenesis found that a dilysine motif mediates EBP binding to coatomer with a conserved function across the Wnt family. We showed that EBP is required for Wnt7a bioactivity when expressed in vivo during regeneration. Overall, our study has elucidated the structural basis and singularity of Wnt secretion on EVs, alternatively to canonical secretion, opening avenues for innovative therapeutic targeting strategies and systemic protein delivery.This work was supported by Frederick Banting and Charles Best Canada Graduate Scholarships–Doctoral Award (to D.D.), Defeat Duchenne Canada (MAR), US National Institutes for Health R01AR044031 (to M.A.R.), Canadian Institutes for Health Research FDN-148387; PJT-183804 (to M.A.R.), Ontario Institute for Regenerative Medicine (to M.A.R.), Stem Cell Network (to M.A.R.), and Ministerio de Ciencia e Innovación (Spain) PID2020-119132GB-100 (to A.H.)
High-Throughput Proteomic Analysis of Human Dermal Fibroblast Response to Different Blood Derivatives: Autologous Topical Serum Derived from Plasma Rich in Growth Factors (PRGF) versus Leukocyte- and Platelet-Rich Plasma (L-PRP)
No full textPlatelet-rich plasma (PRP) is nowadays used in the treatment of different types of cutaneous lesions. However, different compositions can influence clinical outcomes. Among them, the inclusion of leukocytes is controversial. High-throughput proteomics techniques were used to analyze the proteins that are differentially expressed in human dermal fibroblasts (HDFs) after treatment for 24 h with two PRP types, autologous topical serum (Endoret serum—ES) derived from plasma rich in growth factors (PRGF) and leukocyte- and platelet-rich plasma (L-PRP). The identified proteins were then classified by both Gene Ontology and Ingenuity Pathway Analysis. The obtained results show that the compositions of ES and L-PRP differ in such a way that they induce different responses in HDFs. ES-treated HDFs overexpress growth factor-related proteins, leading to protein synthesis, cell proliferation and migration. By contrast, L-PRP treatment induces a response similar to that caused by proinflammatory molecules. These data could explain the contradictory clinical results obtained for the different types of PRP, especially with respect to their leukocyte contents
Embryo-induced alterations in the protein profile of bovine oviductal extracellular vesicles
No full textThe oviduct provides the optimum environment for early embryonic development. Maternal-embryonic communication, which is essential for embryo quality, is mediated partly via extracellular vesicles (EVs). This study aimed to investigate the protein cargo of EVs obtained from the oviductal fluid (OF) of pregnant and cyclic heifers and their implications for maternal-embryonic communication in vivo. Oestrous cycles of crossbred beef heifers were synchronized, following which they were artificially inseminated (pregnant; n=13) or not (cyclic; n= 8) and slaughtered 3.5 days after insemination. The oviduct ipsilateral to the corpus luteum was flushed and the OF was examined to confirm the presence of a 6-8 cell embryo in pregnant animals. OF-EVs were isolated using size exclusion chromatography, concentrated by ultrafiltration, while EVs presence were characterized by flow cytometry using antibodies for specific EV markers (CD63, CD81, and CD44). Proteomic analysis was carried out using nanoLC-MS/MS with spectral counting to identify and quantify the proteins present in the EVs. Five animals from each group were used and statistical analysis was performed using ANOVA for flow cytometry data or T-test for proteomic data, both with a significance level of 5%. Bioinformatic analysis was performed with the DAVID and STRING tools. Flow cytometry analysis confirmed EV presence and no significant differences in EV markers between groups. A total of 1,101 proteins were identified: 5 unique to OF-EVs from cyclic heifers, 611 unique to pregnant heifers, and 485 in common. Among the common proteins, 93 were upregulated and 42 were downregulated in OF-EVs from the pregnant group. Functional enrichment analysis demonstrated that proteins exclusive to pregnant OF-EVs are involved in the Ras and Hippo pathways. Of note, Ras signaling is critical for mouse embryo development at the time of embryonic genome activation, which in cattle occurs in the oviduct during 8- to 16-cells transition. Furthermore, LLGL1, PATJ, and PARD6GB, members of the Hippo pathway exclusively found in pregnant OF-EVs, can regulate cell polarity and establishment of pluripotency. Additional pathways related to unique and upregulated proteins in pregnant OF-EVs include tight junction, cell adhesion molecules, and focal adhesion, which are essential for proper oviductal cell functioning and embryo development. Gene ontology analysis also revealed that upregulated proteins in pregnant OF-EVs, in comparison to EVs from cyclic animals, are associated with the immune response. In conclusion, although our model does not exclude a potential effect of sperm on the OF-EVs in the inseminated group, the study characterized a specific protein signature in OF-EVs from pregnant animals, which is likely due to the interactions established between the mother and the embryo.Financial support: RSGM and MARF received funding for this research from Sao Paulo Research Foundation (FAPESP – Grant 2015/18519-8; 2017/14957-6) and National Council for Scientific and Technological Development (CNPq – Grant 441492/2014-2).Peer reviewe
Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma
No full textApoptosis; Autophagy; Proteasome inhibitorApoptosis; Autofagia; Inhibidor del proteasomaApoptosi; Autofàgia; Inhibidor del proteasomaProtein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy–lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin–proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.This work was supported at early stages by Spanish MINECO, CTQ2011–27874 grant. M.G.-S. is a fellow of the UbiCODE project funded by the EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 765445. M.S.R. and L.C. were also funded by the Institut National du Cancer, France (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie. O.C. is funded by “La Ligue contre le cancer du Gard”. ICFO authors were supported by funding from the Spanish MINECO “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), from Fundació Privada Cellex, Fundación Mig-Puig, from Generalitat de Catalunya CERCA program and from LASERLAB Europe (grant agreement No 871124;). G.R. was financially supported by Fondo de Investigación Sanitaria PI15/00102 and PI18/01383, European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’. G.R. and D.R.G. are members of the Spanish Network of Excellence UBIRed funded by the Spanish Ministry Science, Innovation and Universities (SAF2017-90900-REDT)
Retraction notice to “WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1” (Archives of Biochemistry and Biophysics (2020) 679, (S0003986119306447), (10.1016/j.abb.2019.108223))
No full textPublisher Copyright: © 2025 Elsevier Inc.This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editors-in-Chief. After concerns about figure accuracy in figure 3 of the article were raised, the authors checked their submitted figure and shared the original images with the editor to clarify the inadvertence of the error present in the figure. However, in the editors' opinion, the response did not adequately address the concerns, leading them to decide to retract the article. The corresponding author would like to apologize for any inconvenience caused. The authors do not agree with the retraction and dispute the grounds for it.proofinpres
Extracellular vesicles from thyroid cancer harbor a functional machinery involved in extracellular matrix remodeling
No full textExtracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.Fil: Bravo Miana, Rocío del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Soler, María Florencia. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ceschin, Danilo Guillermo. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Royo, Félix. Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas; EspañaFil: Negretti Borga, Dana Maria. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Azkargorta, Mikel. Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas; EspañaFil: Elortza, Félix. Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas; EspañaFil: Montesinos, Maria del Mar. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Pellizas, Claudia Gabriela. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Falcón-Pérez, Juan Manuel. Centro de Investigación Biomédica En Red de Enfermedades Hepáticas y Digestivas; España. Ikerbasque, Basque Foundation For Science; EspañaFil: Donadio, Ana Carolina. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin
Human tear proteomics and peptidomics in ophthalmology: Toward the translation of proteomic biomarkers into clinical practice
No full textProteostasis disturbances and endoplasmic reticulum stress contribute to polycystic liver disease: New therapeutic targets
No full textBackground & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target.Methods ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro.Results The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis.Conclusions Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy
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