Is Lipid Metabolism of Value in Cancer Research and Treatment? Part I- Lipid Metabolism in Cancer.

For either healthy or diseased organisms, lipids are key components for cellular membranes; they play important roles in numerous cellular processes including cell growth, proliferation, differentiation, energy storage and signaling. Exercise and disease development are examples of cellular environment alterations which produce changes in these networks. There are indications that alterations in lipid metabolism contribute to the development and progression of a variety of cancers. Measuring such alterations and understanding the pathways involved is critical to fully understand cellular metabolism. The demands for this information have led to the emergence of lipidomics, which enables the large-scale study of lipids using mass spectrometry (MS) techniques. Mass spectrometry has been widely used in lipidomics and allows us to analyze detailed lipid profiles of cancers. In this article, we discuss emerging strategies for lipidomics by mass spectrometry; targeted, as opposed to global, lipid analysis provides an exciting new alternative method. Additionally, we provide an introduction to lipidomics, lipid categories and their major biological functions, along with lipidomics studies by mass spectrometry in cancer samples. Further, we summarize the importance of lipid metabolism in oncology and tumor microenvironment, some of the challenges for lipodomics, and the potential for targeted approaches for screening pharmaceutical candidates to improve the therapeutic efficacy of treatment in cancer patients.

Is Lipid Metabolism of Value in Cancer Research and Treatment? Part II: Role of Specialized Pro-Resolving Mediators in Inflammation, Infections, and Cancer.

Acute inflammation is the body's first defense in response to pathogens or injury that is partially governed by a novel genus of endogenous lipid mediators that orchestrate the resolution of inflammation, coined specialized pro-resolving mediators (SPMs). SPMs, derived from omega-3-polyunstaturated fatty acids (PUFAs), include the eicosapentaenoic acid-derived and docosahexaenoic acid-derived Resolvins, Protectins, and Maresins. Herein, we review their biosynthesis, structural characteristics, and therapeutic effectiveness in various diseases such as ischemia, viral infections, periodontitis, neuroinflammatory diseases, cystic fibrosis, lung inflammation, herpes virus, and cancer, especially focusing on therapeutic effectiveness in respiratory inflammation and ischemia-related injuries. Resolvins are sub-nanomolar potent agonists that accelerate the resolution of inflammation by reducing excessive neutrophil infiltration, stimulating macrophage functions including phagocytosis, efferocytosis, and tissue repair. In addition to regulating neutrophils and macrophages, Resolvins control dendritic cell migration and T cell responses, and they also reduce the pro-inflammatory cytokines, proliferation, and metastasis of cancer cells. Importantly, several lines of evidence have demonstrated that Resolvins reduce tumor progression in melanoma, oral squamous cell carcinoma, lung cancer, and liver cancer. In addition, Resolvins enhance tumor cell debris clearance by macrophages in the tumor's microenvironment. Resolvins, with their unique stereochemical structure, receptors, and biosynthetic pathways, provide a novel therapeutical approach to activating resolution mechanisms during cancer progression.

Subchondral bone in knee osteoarthritis: bystander or treatment target?

The subchondral bone is an important structural component of the knee joint relevant for osteoarthritis (OA) incidence and progression once disease is established. Experimental studies have demonstrated that subchondral bone changes are not simply the result of altered biomechanics, i.e., pathologic loading. In fact, subchondral bone alterations have an impact on joint homeostasis leading to articular cartilage loss already early in the disease process. This narrative review aims to summarize the available and emerging imaging techniques used to evaluate knee OA-related subchondral bone changes and their potential role in clinical trials of disease-modifying OA drugs (DMOADs). Radiographic fractal signature analysis has been used to quantify OA-associated changes in subchondral texture and integrity. Cross-sectional modalities such as cone-beam computed tomography (CT), contrast-enhanced cone beam CT, and micro-CT can also provide high-resolution imaging of the subchondral trabecular morphometry. Magnetic resonance imaging (MRI) has been the most commonly used advanced imaging modality to evaluate OA-related subchondral bone changes such as bone marrow lesions and altered trabecular bone texture. Dual-energy X-ray absorptiometry can provide insight into OA-related changes in periarticular subchondral bone mineral density. Positron emission tomography, using physiological biomarkers of subchondral bone regeneration, has provided additional insight into OA pathogenesis. Finally, artificial intelligence algorithms have been developed to automate some of the above subchondral bone measurements. This paper will particularly focus on semiquantitative methods for assessing bone marrow lesions and their utility in identifying subjects at risk of symptomatic and structural OA progression, and evaluating treatment responses in DMOAD clinical trials.

Targeted Therapy of Non-Small Cell Lung Cancer and Liver Cancer: Functional Nanocarriers for the Delivery of Cisplatin and Tissue Factor Pathway Inhibitor-2.

INTRODUCTION: The aim of the study was to construct folic acid-modified PEGylated paramagnetic nanoparticles (MNPs) co-carrying tissue factor pathway inhibitor-2 (TFPI-2) and cisplatin (CDDP), and to study the molecular-targeting and inhibitory effects of the nanocomposite on non-small cell lung cancer (NSCLC) and liver cancer. METHODS: Nanocomposites were prepared using amino-modified iron oxide nanoparticles as carriers, co-loading CDDP and PEGylated FA/TFPI-2. Transmission electron microscopy, UV absorption spectrum, and dynamic light scattering were employed to characterize the morphology, structure, particle size, and zeta potential of the nanocomposite. The phenylenediamine method was used to detect the loading of CDDP, and the CCK-8 assay was used to detect the toxic effect of the nanocomposite on HUVECs, A549, and NCI-H460 cells. In tumor-bearing mice models, the antitumor effects of the nanocomposites were assessed using TUNEL staining (at the molecular level), reverse transcriptase quantitative polymerase chain reaction (at the gene level), hematoxylin and eosin staining (at the cellular level), and the appearance of the mice models. RESULTS: The synthesized FA-MNP/CDDP/TFPI-2 nanocomposite was uniformly dispersed and spherical in shape (approximate diameter: 10 nm). The zeta potential of particles was -9.44 mV, and the average particle size was 25 nm. The loading amount of CDDP was 70.24 µg/mL (23.33%). The nanocomposite was nontoxic to HUVECs, while it showed a favorable inhibitory effect on A549 and NCI-H460 cells. In vivo experiments in mice demonstrated satisfactory imaging properties and therapeutic effects of nanocomposite against liver cancer. DISCUSSION: FA-MNP/CDDP/TFPI-2 may provide insights for the development of new chemotherapeutic drugs.

Application of functional training on physical fitness in basketball / Aplicação do treinamento funcional sobre a aptidão física no basquetebol / Aplicación del entrenamiento funcional en la aptitud física en el baloncesto

ABSTRACT Introduction: Basketball is a predominant competitive sport among the masses. Its practice requires scoring as many points as possible within a specific time limit, manifesting a short-duration, high-intensity intermittent training session. To meet the sport's requirements, the athlete must have ample directional control, rapid changes in speed, and good jumping ability. Objective: Verify the effects of functional training on basketball players aiming to improve explosive power, stability, and agility. Methods: 18 college basketball players were randomly selected as volunteers for the investigation. They were randomly divided into experimental and control groups. Only to the experimental one was functional physical training added during routine training. The trial lasted for eight weeks. All participants were tested for agility, strength, speed, endurance, power, and cartilage repair before and after the experiment. Finally, a comprehensive statistical data analysis was performed. Results: There were significant differences between the experimental group and the control group in push-ups, 3200-meter run, physical acuity detection, 17 sidelines, and running items, and touch height (P<0.05). In the experimental group, there was no significant difference in high school grades (P>0.05). There was significant statistical significance in the supine project, the 3200-meter athletics project, the 17 sideline projects, and the touchdown project (P<0.05). Conclusion: The physical fitness of the experimental group improved significantly after functional physical training. After routine physical training, the control group athletes had significantly positive absolute strength and endurance results. Overall, functional fitness training is superior to conventional fitness training. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: O basquetebol é um esporte de competição predominante entre as massas. Sua prática requer o maior número possível de pontos dentro de um limite de tempo específico, manifestando uma sessão de treinamento intermitente de curta duração e de alta intensidade. Para atender aos requisitos esportivos, é necessário que o atleta tenha um amplo controle direcional, mudanças rápidas de velocidade e boa capacidade de salto. Objetivo: Verificar os efeitos do treinamento funcional em jogadores de basquetebol visando melhorar o poder explosivo, estabilidade e agilidade. Métodos: Foram selecionados aleatoriamente 18 jogadores universitários de basquetebol como voluntários para a investigação. Divididos aleatoriamente em grupos experimental e controle. Apenas ao experimental foi adicionado o treinamento físico funcional durante o treinamento de rotina. O ensaio durou oito semanas. Todos os participantes foram testados quanto a agilidade, força, velocidade, resistência, potência e reparo de cartilagem antes e depois do experimento. Finalmente, efetuou-se uma abrangente análise estatística de dados. Resultados: Verificou-se diferenças significativas entre o grupo experimental e o grupo de controle em flexões, 3200 metros de corrida, detecção de acuidade física, 17 itens de linha lateral e itens de corrida e altura de toque (P<0,05). No grupo experimental, não houve diferença significativa nas notas do ensino médio (P>0,05). Houve significância estatística significativa no projeto de supino, no projeto de atletismo de 3200 metros, nos 17 projetos de linha lateral, e no projeto de retoque (P<0,05). Conclusão: A aptidão física do grupo experimental melhorou significativamente após o treinamento físico funcional. Após o treinamento físico de rotina, os atletas do grupo de controle tiveram resultados significativamente positivos sobre força e resistência absolutas. Em geral, o treinamento de aptidão física funcional é superior ao treinamento de aptidão física convencional. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

Resumen Introducción: El baloncesto es un deporte de competición predominante entre las masas. Su práctica requiere el mayor número posible de puntos dentro de un límite de tiempo específico, manifestando una sesión de entrenamiento intermitente de corta duración y alta intensidad. Para cumplir con los requisitos deportivos, es necesario que el atleta tenga un amplio control direccional, rápidos cambios de velocidad y una buena capacidad de salto. Objetivo: Verificar los efectos del entrenamiento funcional en jugadores de baloncesto con el objetivo de mejorar la potencia explosiva, la estabilidad y la agilidad. Métodos: 18 jugadores de baloncesto universitario fueron seleccionados al azar como voluntarios para la investigación. Se dividieron aleatoriamente en grupos experimentales y de control. Sólo al experimental se le añadió el entrenamiento físico funcional durante el entrenamiento rutinario. La prueba duró ocho semanas. Todos los participantes fueron sometidos a pruebas de agilidad, fuerza, velocidad, resistencia, potencia y reparación del cartílago antes y después del experimento. Por último, se realizó un exhaustivo análisis estadístico de los datos. Resultados: Hubo diferencias significativas entre el grupo experimental y el grupo de control en las flexiones de brazos, la carrera de 3200 metros, la detección de la agudeza física, los 17 elementos de la banda y la carrera y la altura del toque (P<0,05). En el grupo experimental, no hubo diferencias significativas en las calificaciones de la escuela secundaria (P>0,05). Hubo una significación estadística significativa en el proyecto de decúbito supino, el proyecto de 3200 metros de atletismo, los 17 proyectos de banda y el proyecto de touchdown (P<0,05). Conclusión: La aptitud física del grupo experimental mejoró significativamente tras el entrenamiento físico funcional. Tras el entrenamiento físico rutinario, los atletas del grupo de control obtuvieron resultados significativamente positivos en cuanto a fuerza y resistencia absolutas. En general, el entrenamiento de aptitud física funcional es superior al entrenamiento de aptitud física convencional. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris.

Introduction: The industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability. Methods: Wild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed. Results and discussion: The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability. Conclusion: These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

Preliminary comparison of the efficacy of several surgical treatments based on maxillomandibular advancement procedures in adult patients with obstructive sleep apnoea: a systematic review and network meta-analysis.

PURPOSE: To evaluate the efficacy of eight different surgical treatments based on maxillomandibular advancement (MMA), which has emerged in recent years, for adult obstructive sleep apnoea (OSA) patients. METHODS: The literature was searched from January 2010 to May 2020 for studies of adult OSA patients with different types of MMA procedures to perform a network meta-analysis. The outcomes were changes in the apnoea-hypopnoea index (AHI), the lowest pulse oxygen saturation (SpO2 min) and the Epworth Sleepiness Scale (ESS). Treatment hierarchy was summarized according to the rank charts. RESULTS: Eight studies were included and encompassed a total of 227 adult patients diagnosed with OSA. Among them, 225 patients underwent combined surgery or simple MMA surgery, including modified maxillomandibular advancement (MMMA),counterclockwise maxillomandibular advancement (CMMA), drug-induced sleep endoscopy and maxillomandibular advancement (MMA + DISE), transoral robotic surgery and maxillomandibular advancement (MMA + TORS), uvulopalatopharyngoplasty (UPPP), maxillomandibular advancement and uvulopalatopharyngoplasty (MMA + UPPP), uvulopalatopharyngoplasty with uvula preservation and maxillomandibular advancement (MMA + HUPPP); MMA consisting of Le Fort I osteotomy and bilateral inverted-L osteotomy (ILOs), genioplasty and iliac bone grafting; and MMA consisting of Le Fort I osteotomy, bilateral sagittal split ramus osteotomies and genioplasty. The results showed that the most effective surgical treatment is MMA + HUPPP [- 56.79 (WMD); 95% confidence interval (CI): - 113.02 to - 3.33] (P < 0.00001), which was far superior to other approaches. CONCLUSION: MMA combined with HUPPP had the highest efficacy. The MMA consisted of Le Fort I osteotomy, bilateral sagittal split ramus osteotomies and genioplasty; CMMA and MMA + TORS are likely also great choices.

Therapeutic Delivery of Polymeric Tadpole Nanostructures with High Selectivity to Triple Negative Breast Cancer Cells.

Targeted delivery of therapeutic drugs using nanoparticles to the highly aggressive triple negative breast cancer cells has the potential to reduce side effects and drug resistance. Cell entry into triple negative cells can be enhanced by incorporating cell binding receptor molecules on the surface of the nanoparticles to enhance receptor-mediated entry pathways, including clatherin or caveolae endocytosis. However, for highly aggressive cancer cells, these pathways may not be effective, with the more rapid and high volume uptake from macropinocytosis or phagocytosis being significantly more advantageous. Here we show, in the absence of attached cell binding receptor molecules, that asymmetric polymer tadpole nanostructure coated with a thermoresponsive poly(N-isopropylacrylamide) polymer with approximately 50% of this polymer in a globular conformation resulted in both high selectivity and rapid uptake into the triple breast cancer cell line MDA-MB-231. We found that the poly(N-isopropylacrylamide) surface coating in combination with the tadpole's unique shape had an almost 15-fold increase in cell uptake compared to spherical particles with the same polymer coating, and that the mode of entry was most likely through phagocytosis. Delivery of the tadpole attached with doxorubicin (a prodrug, which can be released at pHs < 6) showed a remarkable 10-fold decrease in the IC50 compared to free doxorubicin. It was further observed that cell death was primarily through late apoptosis, which may allow further protection from the body's own immune system. Our results demonstrate that by tuning the chemical composition, polymer conformation and using an asymmetric-shaped nanoparticle, both selectivity and effective delivery and release of therapeutics can be achieved, and such insights will allow the design of nanoparticles for optimal cancer outcomes.

Membrane type 1-matrix metalloproteinase induces epithelial-to-mesenchymal transition in esophageal squamous cell carcinoma: Observations from clinical and in vitro analyses.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is associated with enhanced tumorigenicity in many cancers. A recent study has revealed that MT1-MMP induces epithelial-to-mesenchymal transition (EMT) in prostate and breast cancer cells. However, its role in esophageal squamous cell carcinoma (ESCC) has not been studied. Here, we investigated the role of MT1-MMP in the dissemination of ESCC. Expression of MT1-MMP was detected by immunohistochemistry and tissue microarray in 88 Kazakh ESCC patients. Western blotting was performed to detect endogenous and overexpressed exogenous MT1-MMP in the Eca109 and Eca9706 cell lines, respectively. Transwell assay was used to estimate MT1-MMP-induced invasion and metastasis. EMT-associated proteins were detected by immunohistochemistry and western blotting. The associations between the expression of MT1-MMP and EMT-associated proteins with clinicopathologic parameters were analyzed. Overexpression of MT1-MMP was confirmed in Kazakh ESCC patients. MT1-MMP levels were found to be correlated with the depth of tumor infiltration. MT1-MMP induced EMT in ESCC both in vivo and in vitro, N-cadherin and Vimentin expression was upregulated upon MT1-MMP transfection into cells. However, E-cadherin was found to be downregulated. MT1-MMP-induced EMT led to increase migration and invasion in ESCC cell lines. In conclusion, our results suggest that MT1-MMP promotes ESCC invasion and metastasis.

Distinct structural changes in wild-type and amyloidogenic chicken cystatin caused by disruption of C95-C115 disulfide bond.

Human cystatin C (HCC) amyloid angiopathy (HCCAA) is characterized by tissue deposition of amyloid fibrils in blood vessels, which can lead to recurrent hemorrhagic stroke. Wild-type HCC forms part of the amyloid deposits in brain arteries of elderly people with amyloid angiopathy. A point mutation causing a glutamine to a leucine substitution at residue 68 in the HCC polypeptide chain greatly increases the amyloidogenic propensity of HCC and causes a more severe cerebral hemorrhage and premature death in young adults. In this study, we used molecular dynamics simulations to assess the importance of disulfide bridge formation upon the stability of chicken cystatin and how this may influence the propensity for amyloid formation. We found that disulfide bridge formation between Cys95 and Cys115 in human cystatin played a critical role in overall protein stability. Importantly, Cys95-Cys115 influenced cystatin structure in regions of the protein that play key roles in the protein-folding transitions that occur, which enable amyloid fibril formation. We hypothesized that correct disulfide bridge formation is a critical step in stabilizing cystatin toward its native conformation. Disrupting Cys95-Cys115 disulfide bridge formation within cystatin appears to significantly enhance the amyloidogenic properties of this protein. In addition, by combining in silico studies with our previous experimental results on Eps1, a molecular chaperone of the PDI family, we proposed that age-related HCCAA, may possess a different pathogenic mechanism compared with its amyloidogenic counterpart, the early onset amyloidogenic cystatin-related CAA.

(-)-epigallocatechin-3-gallate inhibits fibrillogenesis of chicken cystatin.

Previous studies have reported that (-)-epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, can bind to unfolded native polypeptides and prevent conversion to amyloid fibrils. To elucidate whether this antifibril activity is specific to disease-related target proteins or is more generic, we investigated the ability of EGCG to inhibit amyloid fibril formation of amyloidogenic mutant chicken cystatin I66Q, a generic amyloid-forming model protein that undergoes fibril formation through a domain swapping mechanism. We demonstrated that EGCG was a potent inhibitor of amyloidogenic cystatin I66Q amyloid fibril formation in vitro. Computational analysis suggested that EGCG prevented amyloidogenic cystatin fibril formation by stabilizing the molecule in its native-like state as opposed to redirecting aggregation toward disordered and amorphous aggregates. Therefore, although EGCG appears to be a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves such inhibition may be specific to the target fibril-forming polypeptide.

TGF-ß1/Smad signaling pathway regulates epithelial-to-mesenchymal transition in esophageal squamous cell carcinoma: in vitro and clinical analyses of cell lines and nomadic Kazakh patients from northwest Xinjiang, China.

Invasion and metastasis are the major causes of death in patients with esophageal squamous cell carcinoma (ESCC). Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-ß1 (TGF-ß1) signaling has been shown to play an important role in EMT. In this study, we investigated how TGF-ß1 signaling pathways contributed to EMT in three ESCC cell lines as well as 100 patients of nomadic ethnic Kazakhs residing in northwest Xinjiang Province of China. In vitro analyses included Western blotting to detect the expression of TGF-ß1/Smad and EMT-associated proteins in Eca109, EC9706 and KYSE150 cell lines following stimulation with recombinant TGF-ß1 and SB431542, a potent inhibitor of ALK5 that also inhibits TGF-ß type II receptor. TGF-ß-activated Smad2/3 signaling in EMT was significantly upregulated as indicated by mesenchymal markers of N-cadherin and Vimentin, and in the meantime, epithelial marker, E-cadherin, was markedly downregulated. In contrast, SB431542 addition downregulated the expression of N-cadherin and Vimentin, but upregulated the expression of E-cadherin. Moreover, the TGF-ß1-induced EMT promoted invasion capability of Eca109 cells. Tumor cells undergoing EMT acquire fibroblastoid-like phenotype. Expressed levels of TGF-ß1/Smad signaling molecules and EMT-associated proteins were examined using immunohistochemical analyses in 100 ESCC tissues of Kazakh patients and 58 matched noncancerous adjacent tissues. The results showed that ESCC tissues exhibited upregulated expression of TGF-ß1/Smad. We also analyzed the relationship between the above proteins and the patients' clinicopathological characteristics. The TGF-ß1/Smad signaling pathway in human Eca109 ESCC cells may carry similar features as in Kazakh ESCC patients, suggesting that TGF-ß1/Smad signaling pathway may be involved in the regulation of EMT in ethnic Kazakh patients with ESCC from Xinjiang, China.

Anticancer activity of binary toxins from Lysinibacillus sphaericus IAB872 against human lung cancer cell line A549.

The inhibitory effect of binary toxic (Bin) protein produced by Lysinibacillus sphaericus IAB872 on cell proliferation of human lung, liver, stomach and cervical tumor cell lines was assessed using MTT assay. The effect of Bin protein on A549 cell proliferation, apoptosis, cell cycle, migration and invasion were examined by MTT assay, Western blotting, Immunocytochemical staining, flow cytometry assay and wound-healing assay. Results showed that Bin protein inhibits proliferation of a range of human cancer cells in vitro. The anti-proliferative effect of Bin is associated with cell apoptosis as a result of an increased ratio of cellular Bax/bcl-2, up-regulated CyclinB1 and down-regulated Cdc25c expression, and its anti-proliferative action was associated with cell cycle arrest in the G2/M-phase. Bin protein could promote apoptosis and inhibit motility and invasion of A549 cancer cells. The anti-proliferative effect of Bin protein was associated with the induction of apoptotic cell death and cell cycle disruption. These results show that Bin protein has the potential to be developed as a chemotherapeutic agent by induction of human tumor cell apoptosis.

Steered molecular dynamics simulation of the binding of the ß2 and ß3 regions in domain-swapped human cystatin C dimer.

The crystal structure of the human cystatin C (hCC) dimer revealed that a stable twofold-symmetric dimer was formed via 3D domain swapping. Domain swapping with the need for near-complete unfolding has been proposed as a possible route for amyloid fibril initiation. Thus, the interesting interactions that occur between the two molecules may be important for the further aggregation of the protein. In this work, we performed steered molecular dynamics (SMD) simulations to investigate the dissociation of the ß2 and ß3 strands in the hCC dimer. The energy changes observed during the SMD simulations showed that electrostatic interactions were the dominant interactions involved in stabilizing the two parts of the dimer during the early stages of SMD simulation, whereas van der Waals (VDW) interactions and electrostatic interactions were equally matched during the latter stages. Furthermore, our data indicated that the two parts of the dimer are stabilized by intermolecular hydrogen bonds among the residues Arg51 (ß2), Gln48 (ß2), Asp65 (ß3), and Glu67 (ß3), salt bridges among the residues Arg53 (ß2), Arg51 (ß2), and Asp65 (ß3), and VDW interactions among the residues Gln48 (ß2), Arg51 (ß2), Glu67 (ß3), Asp65 (ß3), Phe63 (ß3), and Asn61 (ß3). The residues Gln48 (ß2), Arg51 (ß2), Asp65 (ß3) and Glu67 (ß3) appear to be crucial, as they play important roles in both electrostatic and VDW interactions. Thus, the present study determined the key residues involved in the stabilization of the domain-swapped dimer structure, and also provided molecular-level insights into the dissociation process of the hCC dimer.

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant.

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71-Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71-Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71-Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95-Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α-ß interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23.

Steered molecular dynamics simulations on the binding of the appendant structure and helix-ß2 in domain-swapped human cystatin C dimer.

We have performed steered molecular dynamics (SMD) simulations to investigate the dissociation process between the appendant structure (AS) and helix-ß2 in human cystatin C dimer. Energy change during SMD showed that electrostatic interactions, including hydrogen bonds and salt bridges, were the dominant interactions to stabilize the two parts of the dimer. Furthermore, our data indicated that residues, Asn35, Asp40, Ser44, Lys75, and Arg93 play significant roles in the formation of these electrostatic interactions. Docking studies suggested that the interactions between AS and ß2-helix were formed following domain swapping and were responsible for stabilizing the structure of the domain-swapped dimer.

Appendant structure plays an important role in amyloidogenic cystatin dimerization prior to domain swapping.

It has been hypothesized that prior to protein domain swapping, unfolding occurs in regions important for the stability of the native monomeric structure, which probably increases the possibility of intermolecular interaction. In order to explore the detailed information of the important unfolding regions in cystatin prior to domain swapping, 20 ns molecular dynamic simulations were performed at atomic level with typical amyloidogenic chicken cystatin (cC) mutant I66Q monomer under conditions that enable forming amyloid fibrils in biological experiments. Our results showed that I66Q mutant exhibited relatively large secondary structure changes and obvious expanding tendency of hydrophobic core compared to wild-type cC. More importantly, the appendant structure (AS) showed a large displacement and distortion towards the hydrophobic core in amyloidogenic cystatin. The structural analysis on cystatin monomer suggested that structural changes of the AS might make the hydrophobic core expand more easily. In addition, analysis on docking dimer has shown that the distorted AS was favor to intermolecular interactions between two cystatin monomers. Data from an independent theoretical derived algorithm as well as biological experiments also support this hypothesis.

Structural and dynamic properties of a new amyloidogenic chicken cystatin mutant I108T.

Chicken cystatin variant I108T is a mutant in the hydrophobic core of the molecule. It has shown many amyloid-prone characteristics in our previous experimental study. To explore the detailed structural and dynamic properties of the amyloidogenic mutant I108T, 10 ns molecular dynamic simulations of the I108T mutant and wild-type chicken cystatins were performed in this study. Our results suggested that the I108T mutant, which exhibited larger secondary structural fluctuations and hydrophobic core expanding tendency compared with the wild-type chicken cystatin, is a new amyloidogenic form of chicken cystatin, and therefore supported the hypothesis to some extent that site mutations in the hydrophobic core might induce the domain swapping.