Search

Enigma protein, encoded by the PDLIM7 gene, is overexpressed in thyroid cancer in a stage-dependent manner, suggesting a potential involvement in the initiation and progression of thyroid cancer. The Enigma interacts with several cellular pathways, including PI3K/AKT, MDM2, and BMP-1. The Enigma is regulated by microRNAs. Specifically, we showed that the Enigma protein upregulation corresponds to the downregulation of Let-7 family genes. There is limited research on the interactions and regulation of the Enigma with other proteins/genes in thyroid cancer tissues, indicating a gap in current knowledge. Our aim is to establish the Enigma as a biomarker. We also aim to study the interacting partners of the Enigma signaling pathways and their probable miRNA regulation in thyroid cancer progression. Using Western blotting, densitometric analysis, immunoprecipitation (IP), and reverse IP, we detected the protein expression and protein–protein interactions in the corresponding papillary thyroid carcinomas (PTCs). Utilizing real-time qPCR assay and Pearson’s correlation test, we highlighted the correlation between PDLIM7 and Let-7g gene expression in the same tissues. The results showed the differential upregulations of the Enigma protein in different stages of PTCs compared to benign tissues along with AKT, VDR, BMP-1, and MDM2 proteins. Loss of DBP was observed in a subset of PTCs. Strong interactions of the Enigma with PI3K/AKT and MDM2 were noted, along with a weaker BMP-1 interaction. Pearson’s correlation coefficient analysis between PDLIM7 and let-7g gene expression was significant (p < 0.05); however, there was a weak inverse correlation (r = −0.27). The study suggests the potential utility of the PDLIM7-qPCR assay as a biomarker for thyroid cancer. The Enigma’s interactions with key signaling pathways may provide valuable insights into the development of thyroid cancer. The study contributes to understanding the molecular mechanisms involving the Enigma protein in thyroid cancer and highlights its potential as a biomarker.

Enigma protein, encoded by the PDLIM7 gene, is overexpressed in thyroid cancer in a stage-dependent manner, suggesting a potential involvement in the initiation and progression of thyroid cancer. The Enigma interacts with several cellular pathways, including PI3K/AKT, MDM2, and BMP-1. The Enigma is regulated by microRNAs. Specifically, we showed that the Enigma protein upregulation corresponds to the downregulation of Let-7 family genes. There is limited research on the interactions and regulation of the Enigma with other proteins/genes in thyroid cancer tissues, indicating a gap in current knowledge. Our aim is to establish the Enigma as a biomarker. We also aim to study the interacting partners of the Enigma signaling pathways and their probable miRNA regulation in thyroid cancer progression. Using Western blotting, densitometric analysis, immunoprecipitation (IP), and reverse IP, we detected the protein expression and protein–protein interactions in the corresponding papillary thyroid carcinomas (PTCs). Utilizing real-time qPCR assay and Pearson's correlation test, we highlighted the correlation between PDLIM7 and Let-7g gene expression in the same tissues. The results showed the differential upregulations of the Enigma protein in different stages of PTCs compared to benign tissues along with AKT, VDR, BMP-1, and MDM2 proteins. Loss of DBP was observed in a subset of PTCs. Strong interactions of the Enigma with PI3K/AKT and MDM2 were noted, along with a weaker BMP-1 interaction. Pearson's correlation coefficient analysis between PDLIM7 and let-7g gene expression was significant (p < 0.05); however, there was a weak inverse correlation (r = −0.27). The study suggests the potential utility of the PDLIM7-qPCR assay as a biomarker for thyroid cancer. The Enigma's interactions with key signaling pathways may provide valuable insights into the development of thyroid cancer. The study contributes to understanding the molecular mechanisms involving the Enigma protein in thyroid cancer and highlights its potential as a biomarker. [ABSTRACT FROM AUTHOR]

Transforming growth factor ß (TGFß) is required for long-term memory (LTM) for sensitization in Aplysia. When LTM is induced using a two-trial training protocol, TGFß inhibition only blocks LTM when administrated at the second, not the first trial. Here, we show that TGFß acts as a "repetition detector" during the induction of two-trial LTM. Secretion of the biologically inert TGFß proligand must coincide with its proteolytic activation by the Bone morphogenetic protein-1 (BMP-1/Tolloid) metalloprotease, which occurs specifically during trial two of our two-trial training paradigm. This paradigm establishes long-term synaptic facilitation (LTF), the cellular correlate of LTM. BMP-1 application paired with a single serotonin (5HT) pulse induced LTF, whereas neither a single 5HT pulse nor BMP-1 alone effectively did so. On the other hand, inhibition of endogenous BMP-1 activity blocked the induction of two-trial LTF. These results suggest a unique role for TGFß in the interaction of repeated trials: during learning, repeated stimuli engage separate steps of the TGFß cascade that together are necessary for the induction of long-lasting memories. [ABSTRACT FROM AUTHOR]

Bone defects, which can be caused by factors such as trauma, tumor, or osteomyelitis, are clinically common. They lessen the weight a bone is able to bear and cause severe pain to the patient. Although bone transplantation is the gold standard for treating bone defects, it is not suitable for all patients due to its poor availability, risk of spreading disease, and possibility of requiring a secondary surgery. Bone cement as a filler for bone defects can fill any shape of bone defect, and can quickly solidify when injected, providing mechanical strength sufficient for supporting the normal physiological activities of the bone. However, traditional bone cement lacks the ability to induce bone regeneration. Recently, various methods for enhancing the bone regeneration ability of bone cement have been developed, such as adding bone morphogenetic proteins, mesenchymal stem cells, and inorganic substances to bone cement. These methods not only ensure the original biological properties of the bone cement, but also improve the bone cement in terms of its mechanical strength and ability to induce bone regeneration. The aim of this review is to overview the process of bone regeneration, introduce improved bone cement formulations designed to promote bone regeneration, and discuss the clinical application of bone cement and its possibilities for future improvement.

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ± PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively. • C-propeptide mutation phenotype severity is modeled on CPI crystal structure. • Procollagen with C-propeptide mutations localizes to ER lumen, instead of ER membrane. • Mutant C-propeptide pericellular processing is defective, with increased pC-collagen and reduced mature collagen. • BMP-1 processing of mutant C-propeptide in vitro is impaired both with and without enhancer. • Incorporation of pC-collagen disorganizes extracellular matrix. [ABSTRACT FROM AUTHOR]

Purpose: Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis.Method and results: Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells.Conclusions: Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process. [ABSTRACT FROM AUTHOR]

Periostin is a secretory protein with a multi-domain structure, comprising an amino-terminal cysteine-rich EMI domain, four internal FAS 1 domains, and a carboxyl-terminal hydrophilic domain. These adjacent domains bind to extracellular matrix proteins (type I collagen, fibronectin, tenascin-C, and laminin γ2), and BMP-1 that catalyzes crosslinking of type I collagen, and proteoglycans, which play a role in cell adhesion. The binding sites on periostin have been demonstrated to contribute to the mechanical strength of connective tissues, enhancing intermolecular interactions in close proximity and their assembly into extracellular matrix architectures, where periostin plays further essential roles in physiological maintenance and pathological progression. Furthermore, periostin also binds to Notch 1 and CCN3, which have functions in maintenance of stemness, thus opening up a new field of periostin action. [ABSTRACT FROM AUTHOR]

Periostin is a matricellular protein that is composed of a multi-domain structure with an amino-terminal EMI domain, a tandem repeat of four FAS 1 domains, and a carboxyl-terminal domain. These distinct domains have been demonstrated to bind to many proteins including extracellular matrix proteins (Collagen type I and V, fibronectin, tenascin, and laminin), matricellular proteins (CCN3 and βig-h3), and enzymes that catalyze covalent crosslinking between extracellular matrix proteins (lysyl oxidase and BMP-1). Adjacent binding sites on periostin have been suggested to put the interacting proteins in close proximity, promoting intermolecular interactions between each protein, and leading to their assembly into extracellular architectures. These extracellular architectures determine the mechanochemical properties of connective tissues, in which periostin plays an important role in physiological homeostasis and disease progression. In this review, we introduce the proteins that interact with periostin, and discuss how the multi-domain structure of periostin functions as a scaffold for the assembly of interacting proteins, and how it underlies construction of highly sophisticated extracellular architectures. [ABSTRACT FROM AUTHOR]

The two metalloproteases meprin β and bone morphogenetic protein 1 (BMP-1) are both members of the astacin protease family. They share specificity for negatively charged residues around the scissile bond and they are expressed in overlapping compartments of the human body. One important proteolytic substrate they share is pro-collagen I. Ablation of one of the two proteases however leads to different collagen I associated phenotypes in vivo . Over the last years calcium emerged as a regulator for the proteolytic activity of both enzymes. For meprin β a reduction and for BMP-1 an increase in activity was reported under increasing calcium concentrations. Here we revisit different compartments that rely on pro-collagen I maturation and explore the crystal structure of both proteases to highlight possible calcium binding sites. With this we aim to emphasize a to date underestimated regulator that influences both proteases. [ABSTRACT FROM AUTHOR]

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.

Objective CCN family member 2/connective tissue growth factor ( CCN2/ CTGF) is known as an osteogenesis-related molecule and is thought to be implicated in tooth growth. Bone morphogenetic protein-1 ( BMP-1) contributes to tooth development by the degradation of dentin-specific substrates as a metalloprotease. In this study, we demonstrated the correlations between CCN2/ CTGF and BMP-1 in human carious teeth and the subcellular dynamics of BMP-1 in human dental pulp cells. Materials and Methods Expression of CCN2/ CTGF and BMP-1 in human carious teeth was analyzed by immunohistochemistry. BMP-1-induced CCN2/ CTGF protein expression in primary cultures of human dental pulp cells was observed by immunoblotting. Intracellular dynamics of exogenously administered fluorescence-labeled BMP-1 were observed using confocal microscope. Results Immunoreactivities for CCN2/ CTGF and BMP-1 were increased in odontoblast-like cells and reparative dentin-subjacent dental caries. BMP-1 induced the expression of CCN2/ CTGF independently of protease activity in the cells but not that of dentin sialophosphoprotein ( DSPP) or dentin matrix protein-1 ( DMP-1). Exogenously added BMP-1 was internalized into the cytoplasm, and the potent dynamin inhibitor dynasore clearly suppressed the BMP-1-induced CCN2/ CTGF expression in the cells. Conclusion CCN2/ CTGF and BMP-1 coexist beneath caries lesion and CCN2/ CTGF expression is regulated by dynamin-related cellular uptake of BMP-1, which suggests a novel property of metalloprotease in reparative dentinogenesis. [ABSTRACT FROM AUTHOR]

Background Metalloproteases are widely regarded as catalytic enzymes involved in extracellular matrix remodeling in the dental pulp. However, cells internalize several metalloproteases, including matrix metalloprotease (MMP)-2, MMP-3, MMP-9, and MMP-13. Recently, MMP-3 was shown to mediate dental pulp healing as an anti-inflammatory and regenerative factor. Highlight Our previous study showed that MMP-3 stimulates cell migration. This occurs via CCN family member 2/connective tissue growth factor (CCN2/CTGF) expression and secretion in human dental pulp cells, a process dependent on dynamin-related endocytosis and independent of protease activity. Furthermore, we recently revealed that bone morphogenetic protein (BMP)-1, a member of the astacin metalloprotease family, plays a role in reparative dentinogenesis via CCN2/CTGF expression. During this process, the protease activity of BMP-1 is not necessary for CCN2/CTGF expression. These results suggest a new role for metalloproteases in activating signaling cascades required for CCN2/CTGF expression in the dental pulp. Conclusion In this review, we summarize novel roles for metalloproteases, particularly MMP-3 and BMP-1, in dentin–pulp complex repair, with particular emphasis on the effects of CCN2/CTGF expression during regulation of cell migration and tertiary dentinogenesis. [ABSTRACT FROM AUTHOR]

Recent investigations revealed that the "brittle bone" phenotype in osteogenesis imperfecta (OI) is caused not only by dominant mutations in collagen type I genes, but also by recessively inherited mutations in genes responsible for the post-translational processing of type I procollagen as well as for bone formation. The phenotype of patients with mutations in noncollagen genes overlaps with very severe type III and lethal type II OI caused by mutations in collagen genes. Mutations in genes that encode proteins involved in collagen prolyl 3-hydroxylation (P3H1/CRTAP/CyPB) eliminated Pro986 hydroxylation and caused an increase in modification of collagen helix by prolyl 4-hydroxylase and lysyl hydroxylase. However, the importance of these disturbances in the disease pathomechanism is not known. Loss of complex proteins' function as collagen chaperones may dominate the disease mechanism. The latest findings added to the spectrum of OI-causing and collagen-influencing factors other chaperones (HSP47 and FKBP65) and protein BMP-1, which emphasizes the complexity of collagen folding and secretion as well as their importance in bone formation. Furthermore, mutations in genes encoding transcription factor SP7/Osterix and pigment epithelium-derived factor (PEDF) constitute a novel mechanism for OI, which is independent of changes in biosynthesis and processing of collagen. [ABSTRACT FROM AUTHOR]

Abstract: Background: The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin. Scope of review: Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM. Major conclusions: We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13kDa calcium-binding protein via proteolytic cleavage at the dipeptide Ala↓Asp, mediated by BMP-1. The expression of this protease as well as the expression of two key enzymes of the carotinoid metabolism, the β,β-carotene-15,15′-dioxygenase and the retinal dehydrogenase/reductase, were found to be strongly up-regulated by retinoic acid. Hence retinoic acid turned out to be a key factor in skeletogenesis in the most ancient still existing metazoans, the sponges. General significance: It is shown that retinoic acid regulates the formation of the organic cylinder that surrounds the axis of the spicules and enables, as a scaffold, the radial apposition of new silica layers and hence the growth of the spicules. [Copyright &y& Elsevier]

Abstract: Lysyl oxidase (LOX) is a key extracellular enzyme responsible for the post-translational modification of collagens I and III to form mature fibrillar collagen. Increased expression of LOX is associated with fibrosis and cardiac dysfunction, yet little is known about the regulation of LOX in the heart. In this study, the cell signaling pathways responsible for the regulation of LOX expression by transforming growth factor (TGF)-β1 were assessed. Adult cardiac fibroblasts were isolated from male Sprague–Dawley rat hearts by enzymatic digestion. Fibroblasts were grown in DMEM with 10% FBS until approximately 80% confluent, growth arrested for 24h, and then treated with TGF-β1 (0–10ng/ml), in the absence or presence of inhibitors of (1) PI3K (wortmannin), (2) Smad3 (SIS3), (3) p38-MAPK (PD169316), (4) JNK (SP600125) and (5) ERK1/2 (PD98059). TGF-β1 treatment significantly upregulated LOX mRNA and protein expression in cardiac fibroblasts, as well as activity in the cell-conditioned media. Concomitant increases in collagen types I and III, and bone morphogenic protein (BMP-1) expression were found in response to TGF-β1. The increase of LOX protein in response to TGF-β1 was prevented by inhibitors of PI3K, Smad3, p38-MAPK, JNK and ERK1/2. Blockade of PI3K also decreased TGF-β1 induced phosphorylation of Smad3, suggesting that the PI3K/Akt and Smad pathways may be integrated in TGF-β1 signaling. Further studies are warranted to address the regulation of LOX in the normal and diseased heart, and how this critical extracellular enzyme may be targeted for clinical benefit. [Copyright &y& Elsevier]

The article discusses a study which found that astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp). The researchers incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site or a mutant version of that context. They isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. Findings revealed that astacins are expressed by odontoblasts as shown by the developing porcine molars.

Tumour progression in oral squamous cell carcinoma (OSCC) is associated with a reorganisation of extracellular matrix. Laminin-5 (Ln-5) plays an important role for tumour migration and shows an increased expression in areas of direct tumour/stroma interactions. We have previously shown stromal spot like Ln-5/γ2 chain deposits distant from the basement membrane region. In this study we have analysed which cell type is responsible for Ln-5/γ2 chain synthesis in situ. Furthermore, we studied its spatial relation to TGF-β1 as well as the Ln-5 modulating enzymes matrix metalloproteinase (MMP) 2, membrane type-1 (MT1-) MMP and bone morphogenetic protein (BMP-) 1 by different techniques including triple immunofluorescence labelling and in situ hybridisation in OSCC. We found that the stromal spot-like Ln-5 deposits occurred in the invasive front in the vicinity of mesenchymal cells and vessel structures. In particular, not only carcinoma cells but also mesenchymal cells were shown to express the Ln-5/γ2 chain mRNA. Moreover, stromal Ln-5 deposits showed a spatial association with TGF-β1 as well as with MT1-MMP and BMP-1. Based on these findings we suggest that mesenchymal cells contribute to the promotion of tumour cell migration as well as vessel formation in OSCC by providing and organising promigratory Ln-5 fragments. [ABSTRACT FROM AUTHOR]

The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression., (© 2019 Heumüller et al.)