The molecular conformation of silk fibroin regulates osteogenic cell behavior by modulating the stability of the adsorbed protein-material interface

AbstractSilk fibroin (SF) can be used to construct various stiff material interfaces to support bone formation. An essential preparatory step is to partially transform SF molecules from random coils to β-sheets to render the material water insoluble. However, the influence of the SF conformation on osteogenic cell behavior at the material interface remains unknown. Herein, three stiff SF substrates were prepared by varying the β-sheet content (high, medium, and low). The substrates had a comparable chemical composition, surface topography, and wettability. When adsorbed fibronectin was used as a model cellular adhesive protein, the stability of the adsorbed protein-material interface, in terms of the surface stability of the SF substrates and the accompanying fibronectin detachment resistance, increased with the increasing β-sheet content of the SF substrates. Furthermore, (i) larger areas of cytoskeleton-associated focal adhesions, (ii) higher orders of cytoskeletal organization and (iii) more elongated cell spreading were observed for bone marrow-derived mesenchymal stromal cells (BMSCs) cultured on SF substrates with high vs. low β-sheet contents, along with enhanced nuclear translocation and activation of YAP/TAZ and RUNX2. Consequently, osteogenic differentiation of BMSCs was stimulated on high β-sheet substrates. These results indicated that the β-sheet content influences osteogenic differentiation of BMSCs on SF materials in vitro by modulating the stability of the adsorbed protein-material interface, which proceeds via protein-focal adhesion-cytoskeleton links and subsequent intracellular mechanotransduction. Our findings emphasize the role of the stability of the adsorbed protein-material interface in cellular mechanotransduction and the perception of stiff SF substrates with different β-sheet contents, which should not be overlooked when engineering stiff biomaterials.

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Self-Assembled Silk Fibroin-Based Aggregates for Delivery of Camptothecin

Polymers ◽

10.3390/polym13213804 ◽

2021 ◽

Vol 13 (21) ◽

pp. 3804

Author(s):

Javier Pérez Quiñones ◽

Cornelia Roschger ◽

Andreas Zierer ◽

Carlos Peniche-Covas ◽

Oliver Brüggemann

Keyword(s):

Silk Fibroin ◽

Water Soluble ◽

Controlled Delivery ◽

Precipitation Method ◽

Tyrosine Residues ◽

Cycle Arrest ◽

M Phase ◽

The Stability ◽

Β Sheet ◽

Mcf 7

A water-soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol, and testosterone to form SF aggregates for the controlled delivery of the anticancer drug camptothecin (CPT). Elemental analysis and 1H NMR spectroscopy showed a degree of substitution (DS) on SF of 0.4 to 3.8 mol %. Yields of 58 to 71% on vitamins- and testosterone-grafted SF conjugates were achieved. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis–precipitation method, achieving drug contents of 6.3–8.5 wt %. FTIR spectra and DSC thermograms showed that tocopherol- and testosterone-grafted SF conjugates predominantly adopted a β-sheet conformation. After the esterification of tyrosine residues on SF chains with the vitamin or testosterone, the hydrodynamic diameters almost doubled or tripled that of SF. The zeta potential values after esterification increased to about −30 mV, which favors the stability of aggregates in aqueous medium. Controlled and almost quantitative release of CPT was achieved after 6 days in PBS at 37 °C, with almost linear release during the first 8 h. MCF-7 cancer cells exhibited good uptake of CPT-loaded SF aggregates after 6 h, causing cell death and cell cycle arrest in the G2/M phase. Substantial uptake of the CPT-loaded aggregates into MCF-7 spheroids was shown after 3 days. Furthermore, all CPT-loaded SF aggregates demonstrated superior toxicity to MCF-7 spheroids compared with parent CPT. Blank SF aggregates induced no hemolysis at pH 6.2 and 7.4, while CPT-loaded SF aggregates provoked hemolysis at pH 6.2 but not at pH 7.4. In contrast, parent CPT caused hemolysis at both pH tested. Therefore, CPT-loaded SF aggregates are promising candidates for chemotherapy.

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Factors involved in the stability of isolated β-sheets: Turn sequence, β-sheet twisting, and hydrophobic surface burial

Protein Science ◽

10.1110/ps.03520704 ◽

2004 ◽

Vol 13 (4) ◽

pp. 1134-1147 ◽

Cited By ~ 49

Author(s):

Clara M. Santiveri ◽

Jorge Santoro ◽

Manuel Rico ◽

M. Angeles Jiménez

Keyword(s):

Hydrophobic Surface ◽

Β Sheets ◽

The Stability ◽

Β Sheet

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SILK FIBROIN FILMS CRYSTALLIZED BY MULTIWALLED CARBON NANOTUBES

International Journal of Modern Physics B ◽

10.1142/s0217979208047456 ◽

2008 ◽

Vol 22 (09n11) ◽

pp. 1807-1812 ◽

Cited By ~ 4

Author(s):

H.-S. KIM ◽

W.-I. PARK ◽

Y. KIM ◽

H.-J. JIN

Keyword(s):

Electron Microscopy ◽

Carbon Nanotubes ◽

Multiwalled Carbon Nanotubes ◽

Silk Fibroin ◽

Composite Films ◽

Tensile Modulus ◽

Multiwalled Carbon ◽

Regenerated Silk Fibroin ◽

Silk Films ◽

Β Sheet

Silk films prepared from regenerated silk fibroin are normally stabilized by β-sheet formation through the use of solvents (methanol, water etc.). Herein, we report a new method of preparing water-stable films without a β-sheet conformation from regenerated silk fibroin solutions by incorporating a small amount (0.2 wt%) of multiwalled carbon nanotubes (MWCNTs). To extend the biomaterial utility of silk proteins, forming water-stable silk-based materials with enhanced mechanical properties is essential. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the MWCNT-incorporated silk films. The wide-angle X-ray diffraction provided clear evidence of the crystallization of the silk fibroin induced by MWCNT in the composite films without any additional annealing processing. The tensile modulus and strength of the composite films were improved by 108% and 51%, respectively, by the incorporation of 0.2 wt% of MWCNTs, as compared with those of the pure silk films. The method described in this study will provide an alternative means of crystallizing silk fibroin films without using an organic solvent or blending with any other polymers, which may be important in biomedical applications.

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Fabrication and Characterization of Vitamin E TPGS Loaded Silk Fibroin/Hyaluronic Acid Nanofibrous Scaffolds

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.721.274 ◽

2013 ◽

Vol 721 ◽

pp. 274-277

Author(s):

Li Li Ji ◽

Qiao Ling Li ◽

Zeng Hu Yang ◽

Wei Jing Hu ◽

Kui Hua Zhang

Keyword(s):

Hyaluronic Acid ◽

Vitamin E ◽

Silk Fibroin ◽

Ethanol Vapor ◽

Random Coil ◽

Burst Release ◽

Nanofibrous Scaffolds ◽

Vitamin E Tpgs ◽

Polyethylene Glycol 1000 ◽

Β Sheet

Vitamin E d-alpha-tocopheryl polyethylene glycol 1000 succinate (VE TPGS) loaded silk fibroin (SF)/ hyaluronic acid (HA) nanofibrous scaffolds were fabricated by means of electrospinning to biomimic the natural extracellular matrix. Scanning electronic microscopy (SEM) results indicated that electrospun VE TPGS loaded SF/HA nanofibers were ribbon-shaped, the width of nanofibers decreased slightly with the addition of VE TPGS to SF/HA blended solutions. Fourier transform infrared (FTIR) spectroscopy and Wide-angle X-ray diffraction (WAXD) curves revealed that VE TPGS did not induce SF conformation from random coil to β-sheet. SF conformation converted from random coil to β-sheet after being treated with 75% ethanol vapor. In vitro release studies confirmed VE TPGS had no obvious burst release and present good release behavior.

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Correlation between composition and activity of gold nanoparticle conjugates with streptococcal protein G

Biointerface Research in Applied Chemistry ◽

10.33263/briac102.988992 ◽

2020 ◽

Vol 10 (2) ◽

pp. 4988-4992

Keyword(s):

Dissociation Constant ◽

Equilibrium Constants ◽

Gold Surface ◽

Binding Activity ◽

Protein G ◽

Binding Ability ◽

Adsorbed Protein ◽

Streptococcal Protein G ◽

The Stability ◽

Nanoparticle Complex

The composition of the conjugates of gold nanoparticles with streptococcal protein G was studied using fluorescence spectroscopy. The method for determining the composition is based on measuring the intrinsic fluorescence of tryptophan as part of the protein. The equilibrium constants of protein binding by the gold surface were determined using the Sketchard method. An increase in the dissociation constant of the protein–nanoparticle complex for increasing the amount of bound protein was demonstrated, and a relationship was established between the stability of the conjugates, their antigen-binding activity, and the dissociation constant. The effectiveness of the conjugates of different compositions in immunochromatographic assay of specific antibodies against the lipopolysaccharide antigen of Brucella abortus was compared. The binding ability of the conjugates increased along with the amount of protein G to ~200 molecules per nanoparticle. A further increase in the amount of adsorbed protein led to a deterioration in the functional activity of the conjugates.

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Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718177115 ◽

2018 ◽

Vol 115 (8) ◽

pp. 1925-1930 ◽

Cited By ~ 45

Author(s):

Carlos Pardo-Pastor ◽

Fanny Rubio-Moscardo ◽

Marina Vogel-González ◽

Selma A. Serra ◽

Alexandros Afthinos ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

Nuclear Translocation ◽

Focal Adhesions ◽

Leading Edge ◽

Force Transmission ◽

Cellular Processes ◽

Fyn Kinase ◽

Downstream Effector ◽

Metastatic Cells

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

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Multifaceted role of tensins in cancer

10.7287/peerj.preprints.27990 ◽

2019 ◽

Author(s):

Abdulaziz Alfahed ◽

Teresa P Raposo ◽

Mohammad Ilyas

Keyword(s):

Cancer Biology ◽

Focal Adhesions ◽

Adaptor Proteins ◽

Therapeutic Strategies ◽

Cell Behavior ◽

Surrounding Environment ◽

Signaling Mechanisms ◽

Different Types ◽

Biochemical Signals

Tensins are structural adaptor proteins localized at focal adhesions. Tensins can act as mechanosensors and participate in the transduction of biochemical signals from the extracellular matrix to the cytoskeleton, acting as an interface able to alter cell behavior in responses to changes in their surrounding environment. This review aims to provide a concise summary of the main functions of the four known tensins in cell and cancer biology, their homology and recently unveiled signaling mechanisms. We focus specifically on how tensin 4 (TNS4/Cten) may contribute to cancer both as an oncogene supporting metastasis and as tumour suppressor in different types of tissue. A better understanding of the cancer mechanistics involving tensins may provide the rationale for development of specific therapeutic strategies.

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Synergic adhesive chemistry-based fabrication of BMP-2 immobilized silk fibroin hydrogel functionalized with hybrid nanomaterial to augment osteogenic differentiation of rBMSCs for bone defect repair

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2021.09.036 ◽

2021 ◽

Author(s):

Peng Shen ◽

Yi-Rong Wang ◽

Li Miao ◽

Hou-Zhuo Luo ◽

Qian Liu ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Bone Defect ◽

Silk Fibroin ◽

Hybrid Nanomaterial ◽

Bone Defect Repair ◽

Defect Repair

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HP1γ Sensitizes Cervical Cancer Cells to Cisplatin through the Suppression of UBE2L3

International Journal of Molecular Sciences ◽

10.3390/ijms21175976 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5976 ◽

Cited By ~ 1

Author(s):

Sang Ah Yi ◽

Go Woon Kim ◽

Jung Yoo ◽

Jeung-Whan Han ◽

So Hee Kwon

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Nuclear Export ◽

Nuclear Translocation ◽

Leptomycin B ◽

P53 Signaling ◽

Cervical Cancer Cells ◽

Ubiquitin Conjugating Enzyme ◽

The Stability

Cisplatin is the most frequently used agent for chemotherapy against cervical cancer. However, recurrent use of cisplatin induces resistance, representing a major hurdle in the treatment of cervical cancer. Our previous study revealed that HP1γ suppresses UBE2L3, an E2 ubiquitin conjugating enzyme, thereby enhancing the stability of tumor suppressor p53 specifically in cervical cancer cells. As a follow-up study of our previous findings, here we have identified that the pharmacological substances, leptomycin B and doxorubicin, can improve the sensitivity of cervical cancer cells to cisplatin inducing HP1γ-mediated elevation of p53. Leptomycin B, which inhibits the nuclear export of HP1γ, increased cisplatin-dependent apoptosis induction by promoting the activation of p53 signaling. We also found that doxorubicin, which induces the DNA damage response, promotes HP1γ-mediated silencing of UBE2L3 and increases p53 stability. These effects resulted from the nuclear translocation and binding of HP1γ on the UBE2L3 promoter. Doxorubicin sensitized the cisplatin-resistant cervical cancer cells, enhancing their p53 levels and rate of apoptosis when administered together with cisplatin. Our findings reveal a therapeutic strategy to target a specific molecular pathway that contributes to p53 degradation for the treatment of patients with cervical cancer, particularly with cisplatin resistance.

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