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The cell–biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7 days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results. ; Funding for open access charge: CRUE-Universitat Jaume I ; This work was supported by Ministerio Ciencia e Innovación [PID2020-113092RB-C21]; Generalitat Valenciana [GRISOLIAP/2018/091, APOSTD/2020/036, PROMETEO/2020/069]; Universitat Jaume I [UJI-B2021-25]; and Basque Government [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, José Ortega, José Miguel Pedra and Iraide Escobés for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for making the titanium discs.

[EN] Melatonin (MLT) is widely known for regulating the circadian cycles and has been studied for its role in bone regeneration and inflammation. Its application as a coating for dental implants can condition the local micro environment, affecting protein deposition on its surface and the cellular and tissue response. Using sol-gel coatings as a release vehicle for MLT, the aim of this work was to assess the potential of this molecule in improving the osseointegration and inflammatory responses of a titanium substrate. The materials obtained were physicochemically characterized (scanning electron microscopy, contact angle, roughness, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, Si release, MLT liberation, and degradation) and studied in vitro with MC3T3-E1 osteoblastic cells and RAW264.7 macrophage cells. Although MLT application led to an increased gene expression of RUNX2 and BMP2 in 10MTL, it did not improve ALP activity. On the other hand, MLT-enriched sol-gel materials presented potential effects in the adsorption of proteins related to inflammation, coagulation and angiogenesis pathways depending on the dosage used. Using LC-MS/MS, protein adsorption patterns were studied after incubation with human serum. Proteins related to the complement systems (CO7, IC1, CO5, CO8A, and CO9) were less adsorbed in materials with MLT; on the other hand, proteins with functions in the coagulation and angiogenesis pathways, such as A2GL and PLMN, showed a significant adsorption pattern. ; This work was supported by MINECO [MAT MAT2017-86043-R; RTC-2017-6147-1], Universitat Jaume I under [UJI-B2017-37; POSDOC/2019/28], Generalitat Valenciana [GRISOLIAP/2018/091], University of the Basque Country under [UFI11/56] and Basque Government under [PRE_2017_2_0044]. CIC bioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County; The ProteoRed-ISCIII (Grant PRB3 IPT17/0019); CIBERehd Network and Severo ...

[EN] Background Calcium (Ca) is a well-known element in bone metabolism and blood coagulation. Here, we investigate the link between the protein adsorption pattern and the in vivo responses of surfaces modified with calcium ions (Ca-ion) as compared to standard titanium implant surfaces (control). We used LC-MS/MS to identify the proteins adhered to the surfaces after incubation with human serum and performed bilateral surgeries in the medial section of the femoral condyles of 18 New Zealand white rabbits to test osseointegration at 2 and 8 weeks post-implantation (n=9). Results Ca-ion surfaces adsorbed 181.42 times more FA10 and 3.85 times less FA12 (p 0.001), which are factors of the common and the intrinsic coagulation pathways respectively. We also detected differences in A1AT, PLMN, FA12, KNG1, HEP2, LYSC, PIP, SAMP, VTNC, SAA4, and CFAH (p 0.01). At 2 and 8 weeks post-implantation, the mean bone implant contact (BIC) with Ca-ion surfaces was respectively 1.52 and 1.25 times higher, and the mean bone volume density (BVD) was respectively 1.35 and 1.13 times higher. Differences were statistically significant for BIC at 2 and 8 weeks and for BVD at 2 weeks (p 0.05). Conclusions The strong thrombogenic protein adsorption pattern at Ca-ion surfaces correlated with significantly higher levels of implant osseointegration. More effective implant surfaces combined with smaller implants enable less invasive surgeries, shorter healing times, and overall lower intervention costs, especially in cases of low quantity or quality of bone. ; This work was supported by Universitat Jaume I under [POSDOC/2019/28], Generalitat Valenciana [GRISOLIAP/2018/091], University of the Basque Country under [UFI11/56], and Basque Government under [PRE_2017_2_0044]. CIC bioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County; The ProteoRed-ISCIII (Grant PRB3 IPT17/0019); and CIBERehd Network and Severo Ochoa Grant (SEV-2016-0644). ...

[EN] Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces. ; This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091; APOSTD/2020/036, PROMETEO/2020/069], Universitat Jaume I under [ UJI-B2017-37], the University of the Basque Country under [GIU18/189] and Basque Government under [PRE_2017_2_0044]. The authors would like to thank Raquel Oliver, Jose Ortega and Iraide Escobes for their valuable technical assistance. ; Romero-Gavilán, F.; Cerqueira, A.; Anitua, E.; Tejero, R.; García-Arnáez, I.; Martínez-Ramos, C.; Ozturan, S. (2021). Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications. JBIC Journal of Biological ...

Incluye material complementario ; The Human Proteome Project (HPP) consortium aims to functionally characterize the dark proteome. On the basis of the relevance of olfaction in early neurodegeneration, we have analyzed the dark proteome using data mining in public resources and omics data sets derived from the human olfactory system. Multiple dark proteins localize at synaptic terminals and may be involved in amyloidopathies such as Alzheimer's disease (AD). We have characterized the dark PITH domain-containing protein 1 (PITHD1) in olfactory metabolism using bioinformatics, proteomics, in vitro and in vivo studies, and neuropathology. PITHD1-/- mice exhibit olfactory bulb (OB) proteome changes related to synaptic transmission, cognition, and memory. OB PITHD1 expression increases with age in wild-type (WT) mice and decreases in Tg2576 AD mice at late stages. The analysis across 6 neurological disorders reveals that olfactory tract (OT) PITHD1 is specifically upregulated in human AD. Stimulation of olfactory neuroepithelial (ON) cells with PITHD1 alters the ON phosphoproteome, modifies the proliferation rate, and induces a pro-inflammatory phenotype. This workflow applied by the Spanish C-HPP and Human Brain Proteome Project (HBPP) teams across the ON-OB-OT axis can be adapted as a guidance to decipher functional features of dark proteins. Data are available via ProteomeXchange with identifiers PXD018784 and PXD021634. ; This work was funded by grants from the Spanish Ministry of Science Innovation and Universities (ref. PID2019-110356RB-I00 to JFI and ES) and Department of Economic and Business Development from Government of Navarra (ref. 0011-1411-2020-000028) to ES and from MEXT-JSPS 17K08884 and Takeda Science Foundation to IO. The Proteomics Unit of Navarrabiomed is a member of Proteored (PRB3-ISCIII) and is supported by grant PT17/0019/009 to JFI, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER. MLM was supported by a postdoctoral fellowship from the Public University of Navarra (UPNA).

The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code. ; We are thankful to Iraide Escobes for her work in the Proteomics Platform at CIC bioGUNE and Arnoud de Ru in the Center for Proteomics and Metabolomics at the LUMC. O.B.-G., F.T., R.B. and A.C.O.V. acknowledge funding by the grant 765445-EU (UbiCODE Program). R.B. acknowledges funding by grants BFU2017-84653-P and PID2020-114178GB-I00 (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), SAF2017-90900-REDT (UBIRed Program) and IT1165-19 (Basque Country Government). Additional support was provided by the Department of Industry, Tourism, and Trade of the Basque Country Government (Elkartek Research Programs) and by the Innovation Technology Department of the Bizkaia County. VM acknowledges FPI grant PRE2018-086230 (MINECO/FEDER, EU). F.E. is at Proteomics Platform, member of ProteoRed-ISCIII (PT13/0001/0027). F.E. and A.M.A. acknowledge CIBERehd. A.C. acknowledges the Basque Department of education (IKERTALDE IT1106-16), the MCIU ...

Primary cilia are sensory organelles crucial for cell signaling during development and organ homeostasis. Cilia arise from centrosomes and their formation and function is governed by numerous factors. Through our studies on Townes-Brocks Syndrome (TBS), a rare disease linked to abnormal cilia formation in human fibroblasts, we uncovered the leucine-zipper protein LUZP1 as an interactor of truncated SALL1, a dominantly-acting protein causing the disease. Using TurboID proximity labeling and pulldowns, we show that LUZP1 associates with factors linked to centrosome and actin filaments. Here, we show that LUZP1 is a cilia regulator. It localizes around the centrioles and to actin cytoskeleton. Loss of LUZP1 reduces F-actin levels, facilitates ciliogenesis and alters Sonic Hedgehog signaling, pointing to a key role in cytoskeleton-cilia interdependency. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Together with other factors, alterations in LUZP1 may be contributing to TBS etiology. ; Grants which have funded this research: BFU2017-84653-P (MINECO/FEDER, EU) SEV-2016-0644 (Severo Ochoa Excellence Program) 765445-EU (UbiCODE Program) SAF2017-90900-REDT (UBIRed Program) IT634-13 (Basque Country Government)

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo. ; Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (SAF2015-65327-R and RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB, AGL2017-86757-R to LA, SAF2017-87301-R to MLMC, SAF2015-64111-R to AP, SAF2015-73549-JIN to HR), Instituto de Salud Carlos III (PIE13/0004 to AP), the Basque Government Department of Health (2015111117 to LA), the Basque Foundation for Innovation and Health Research (BIOEF), through the EiTB Maratoia grant BIO15/CA/016/BS to MLMC, the regional Government of Andalusia co-funded by CEC and FEDER funds (Proyectos de Excelencia P12-CTS-2232) and Fundación Domingo Martínez (to AP). LA is supported by the Ramon y Cajal program (RYC-2013-13666). DB, MMR and TMM are recipients of MCIU FPI fellowships. ACG and AP are recipients of fellowships form the Basque Government. APC is a recipient of a fellowship from the University of the Basque Country. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia Province and the CIBERehd network. DB and JA are supported by a grant from the Jesús de Gangoiti Barrera Foundation.