Cytoskeleton-related Proteins Research Articles

Temporal Assessment of Protein Stability in Dried Blood Spots.

The use of protein biomarkers in blood for clinical settings is limited by the cost and accessibility of traditional venipuncture sampling. The dried blood spot (DBS) technique offers a less invasive and more accessible alternative. However, protein stability in DBS has not been well evaluated. Herein, we deployed a quantitative LC-MS/MS system to construct proteomic atlases of whole blood, DBSs, plasma, and blood cells. Approximately 4% of detected proteins' abundance was significantly altered during blood drying into blood spots, with overwhelming disturbances in cytoplasmic fraction. We also reported a novel finding suggesting a decrease in the level of membrane/cytoskeletal proteins (SLC4A1, RHAG, DSC1, DSP, and JUP) and an increase in the level of proteins (ATG3, SEC14L4, and NRBP1) related to intracellular trafficking. Furthermore, we identified 19 temporally dynamic proteins in DBS samples stored at room temperature for up to 6 months. There were three declined cytoskeleton-related proteins (RDX, SH3BGRL3, and MYH9) and four elevated proteins (XPO7, RAN, SLC2A1, and SLC29A1) involved in cytoplasmic transport as representatives. The instability was governed predominantly by hydrophilic proteins and enhanced significantly with an increasing storage time. Our analyses provide comprehensive knowledge of both short- and long-term storage stability of DBS proteins, forming the foundation for the widespread use of DBS in clinical proteomics and other analytical applications.

Impact and mechanisms of drag-reducing polymers on shear stress regulation in pulmonary hypertension.

Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear. This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2+] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2+] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways. Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2+] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways. This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

Hsp90 Promotes Gastric Cancer Cell Metastasis and Stemness by Regulating the Regional Distribution of Glycolysis-Related Metabolic Enzymes in the Cytoplasm.

Heat-shock protein 90 (Hsp90) plays a crucial role in tumorigenesis and tumor progression; however, its mechanism of action in gastric cancer (GC) remains unclear. Here, the role of Hsp90 in GC metabolism is the focus of this research. High expression of Hsp90 in GC tissues can interact with glycolysis, collectively affecting prognosis in clinical samples. Both in vitro and in vivo experiments demonstrate that Hsp90 is able to regulate the migration and stemness properties of GC cells. Metabolic phenotype analyses indicate that Hsp90 influences glycolytic metabolism. Mechanistically, Hsp90 interacts with glycolysis-related enzymes, forming multienzyme complexes to enhance glycolysis efficiency and yield. Additionally, Hsp90 binds to cytoskeleton-related proteins, regulating the regional distribution of glycolytic enzymes at the cell margin and lamellar pseudopods. This effect could lead to a local increase in efficient energy supply from glycolysis, further promoting epithelial-mesenchymal transition (EMT) and metastasis. In summary, Hsp90, through its interaction with metabolic enzymes related to glycolysis, forms multi-enzyme complexes and regulates regional distribution of glycolysis by dynamic cytoskeletal adjustments, thereby promoting the migration and stemness of GC cells. These conclusions also support the potential for a combined targeted approach involving Hsp90, glycolysis, and the cytoskeleton in clinical therapy.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: A Novel Role of Actin Cytoskeleton in Disease Progression

Background: Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Methods: To uncover potential biomarkers related to disease development, proteomic profiling using SOMAscan proteomic platform was conducted on Qatari individuals with prehypertension and hypertension. In this study, proteomic data collected from the Qatar Biobank were analyzed to compare participants with prehypertension, hypertension, and normal blood pressure. Statistical analysis was performed to identify altered protein expression between the study groups and to elucidate the biological pathways contributing to this disease. Results: The results revealed a cluster of proteins, including the SRC family, CAMK2B and CAMK2D, TEC, GSK3, VAV, and RAC, that were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤ -1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling, a crucial pathological event involved in hypertension progression. Conclusion: Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease. This study was made possible through the support of Qatar University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomic Analysis of Prehypertensive and Hypertensive Patients: Exploring the Role of the Actin Cytoskeleton.

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation

AbstractThe inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody–based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain–containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Proteomic profiling analysis of human endometrium in women with unexplained recurrent spontaneous abortion

Unexplained recurrent spontaneous abortion (URSA) seriously affects female reproductive health, causing a great burden to patients both physically and mentally. Endometrial decidualization plays an important role in pregnancy, and impaired decidualization is an essential cause of URSA, but the cause of the damage is still poorly understood. This study aimed to reveal the pathogenesis of URSA by analyzing the differential protein expression profiles in the decidual tissue of patients with recurrent abortion compared to those with normal pregnancy. Morphological analysis revealed abnormal decidualization of endometrial tissue in patients with URSA. Quantitative proteomics analysis showed that a total of 146 differentially expressed proteins were identified between the two groups, among which 95 proteins were downregulated and 51 proteins were upregulated. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that the protein expression profile and signaling pathways of endometrium in patients with URSA changed significantly, and cytoskeleton remodeling and morphological transformation disorders were associated with abortion induced by incomplete decidualization. Meanwhile, transcription factors analysis showed that the 3 most affected families were zf-C2H2, MYB and HMG. Therefore, our study may provide a basis for searching for potential markers of decidualization injury. SignificanceAt present, there are still about 50% of RSA patients with unknown causes, which brings great difficulties and blindness to clinical diagnosis and treatment.The limited proteomic studies on URSA further contribute to the lack of understanding in this field. However, in this study, the focus was on proteomic profiling analysis of the human endometrium in URSA patients compared to normal women. The findings revealed that cytoskeletal remodeling disorder is a significant contributor to the failure of decidualization in URSA patients. This insight highlights the potential role of cytoskeleton-related proteins in the pathogenesis of URSA, providing valuable information for further research and potential therapeutic interventions.

Effects of microcystin on protein profile in hepatopancreas of Litopenaeus vannamei.

For intensive aquaculture in freshwater ponds, microcystin (MC-LR) generated from cyanobacterial blooms is one of the bottlenecks for the healthy and sustainable development of shrimp aquaculture industry. In this study, we measured the MC-LR content in the hepatopancreas and muscles of Litopenaeus vannamei stressed by MC-LR, and analyzed protein expression in the hepatopancreas using DIA high-throughput proteomics technology. The results showed that MC-LR content in the hepatopancreas and muscles reached the highest at 1 h after MC-LR injection, which was (6.12±0.45) μg·kg-1 and (5.00±0.19) μg·kg-1, respectively. Then, it decreased gra-dually, with that in the hepatopancreas being significantly higher than in muscles. We identified 820 differential expressed proteins, including 586 up-regulated and 234 down-regulated ones. Results of bioinformatics analysis showed that MC-LR stress significantly affected immune-related pathways such as lysosome, RIG-Ⅰ receptor signals and interleukin-2. It also altered energy metabolisms including citrate cycle, metabolism of starch and sucrose, and interconversion of pentose and glucoronate, which in turn led to the disorder of carbohydrate metabolism. In addition, MC-LR significantly upregulated 19 cytoskeleton-related blood shadow proteins and damaged the hepatopancreas cytoskeleton. It was concluded that MC-LR mainly affected the physiological processes associated with immunity, energy metabolism, and cytoskeleton in the hepatopancreas of L. vannamei.

Zhuidu Formula suppresses the migratory and invasive properties of triple-negative breast cancer cells via dual signaling pathways of RhoA/ROCK and CDC42/MRCK

Ethnopharmacological relevanceZhuidu Formula (ZDF) is composed of triptolide, cinobufagin and paclitaxel, which are the active ingredients of Tripterygium wilfordii Hook. F, dried toad skin and Taxus wallichiana var. chinensis (Pilg) Florin, respectively. Modern pharmacological studies show that triptolide, cinobufagin, and paclitaxel are well-known natural compounds that exert anti-tumor effects by interfering with DNA synthesis, inducing tumor cell apoptosis, and inhibiting the dynamic balance of the tubulin. However, the mechanism by which the three compounds inhibit triple-negative breast cancer (TNBC) metastasis is unknown. ObjectiveThe objective of this investigation was to examine the inhibitory essences of ZDF on the metastasis of TNBC and elucidate its potential mechanism. Materials and methodsCell viability of triptolide (TPL), cinobufagin (CBF), and paclitaxel (PTX) on MDA-MB-231 cells was assessed employing a CCK-8 assay. The drug interactions of the three drugs on MDA-MB-231 cells were determined in vitro utilizing the Chou-Talalay method. MDA-MB-231 cells were identified for migration, invasion and adhesion in vitro through the implementation of the scratch assay, transwell assay and adhesion assay, respectively. The formation of cytoskeleton protein F-actin was detected by immunofluorescence assay. The expressions of MMP-2 and MMP-9 in the supernatant of the cells were determined by ELISA analysis. The Western blot and RT-qPCR were employed to explore the protein expressions associated with the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. The anti-tumor efficacy of ZDF in vivo and its preliminary mechanism were investigated in the mouse 4T1 TNBC model. ResultsThe results demonstrated that ZDF could significantly reduce the viability of the MDA-MB-231 cell, and the combination index (CI) values of actual compatibility experimental points were all less than 1, demonstrating a favorable synergistic compatibility relationship. It was found that ZDF reduces RhoA/ROCK and CDC42/MRCK dual signaling pathways, which are responsible for MDA-MB-231cell migration, invasion, and adhesion. Additionally, there has been a significant reduction in the manifestation of cytoskeleton-related proteins. Furthermore, the expression levels of RhoA, CDC42, ROCK2, and MRCKβ mRNA and protein were down-regulated. ZDF significantly decreased the protein expressions of vimentin, cytokeratin-8, Arp2 and N-WASP, and inhibited actin polymerization and actomyosin contraction. Furthermore, MMP-2 and MMP-9 levels in the high-dose ZDF group were decreased by 30% and 26%, respectively. ZDF significantly reduced the tumor volume and protein expressions of ROCK2 and MRCKβ in tumor tissues without eliciting any perceptible alterations in the physical mass of the mice, and the reduction was more pronounced than that of the BDP5290 treated group. ConclusionThe current investigation demonstrates that ZDF exhibits a proficient inhibitory impact on TNBC metastasis by regulating cytoskeletal proteins through the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. Furthermore, the findings indicate that ZDF has significant anti-tumorigenic and anti-metastatic characteristics in breast cancer animal models.

Abstract LB019: DUSP4 overexpression exerts anti-tumor effects in vivo in pancreatic cancer xenografts in nu/nu mice

Abstract Pancreatic cancer (PC) remains one of the most lethal of all human malignancies, being highly aggressive and difficult to treat if diagnosed at late stages. Therefore, it is important to dissect molecular mechanisms implicated in PC pathophysiology. Dual specificity phosphatase-4 (DUSP4) is a phosphatase known to negatively regulate the mitogen-activated protein (MAP) kinase family (MAPK/ERK, SAPK/JNK, P38), which are involved in multiple cancers, including PC. Previously, using tissue microarray containing a variety of pancreatic cancers and normal pancreatic tissues, we found that DUSP4 was significantly downregulated in human PC. We also demonstrated that forced overexpression of DUSP4 resulted in anti-proliferative responses in MIA PaCa-2 human PC cells (Cancer Res 2016; 76 (14_Supplement): 3667). Here, we expanded our study to determine, i) the effects of DUSP4 overexpression on tumorigenicity in vivo, and ii) the molecular mechanisms associated with DUSP4 overexpression-mediated anti-proliferative responses. MIA PaCa-2 cells were stably transfected with DUSP4 overexpression (OE) plasmid or its empty vector (pCMV6) control, followed by subcutaneous implantation into nu/nu mice (n=8 each group). Tumor growth was measured weekly for 6 weeks followed by excision of tumors and molecular analyses. We found significant reduction in tumor volume and weight in DUSP4 OE group. Further, we found marked reduction in p-ERK1/2 and p-P38 proteins in tumors from DUSP4 OE group. To determine the global mechanisms of DUSP4, we analyzed DUSP4-overexpressing MIA PaCa-2 cell lysates using quantitative global proteomics. Overall, 1678 proteins were identified with ≥2 unique peptides. Out of these, 54 proteins were significantly modulated ≥1.6-fold. Using Ingenuity Pathway Analysis (IPA), we found the association of modulated proteins with increased apoptosis and inhibited invasive and malignant tumors. The top five modulated proteins identified in response to DUSP4 OE were ADD3, PDLIM1, COL2A1, SLC20A1 and CALB2, with the first three being upregulated and the remaining two downregulated. Based on published studies in other cancers, these data support the tumor suppressor role of DUSP4 in PC since i) the loss of ADD3 and PDLIM1 (both cytoskeleton-related proteins) have been found to promote tumor growth, ii) higher expression of COL2A1 has been associated with delayed tumor recurrence, iii) higher expression of SLC20A1 (sodium-phosphate symporter) and CALB2 (calcium-binding protein) are known to be associated with cancer progression. The specific roles of these proteins in PC are not well known, providing us novel opportunities for future investigations. Overall, our data support the tumor suppressor role of DUSP4 in PC and warrants further research to validate our findings. Citation Format: Chandra K. Singh, Mary A. Ndiaye, Gagan Chhabra, Minakshi Nihal, Nihal Ahmad. DUSP4 overexpression exerts anti-tumor effects in vivo in pancreatic cancer xenografts in nu/nu mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB019.

Abstract 2017: A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition

Abstract Comprehensive proteomic profiling of plasmas collected before and after neo-adjuvant treatment for gastric cancer (GC) yielded 522 proteins exhibiting higher pre-treatment levels compared to healthy controls and among them, 345 proteins decreased after the treatment. Increased levels of cytoskeleton-related proteins were manifest in the plasmas of GC patients. Cytoskeletal proteins are known to play integral roles in modulating epithelial plasticity by altering mechanical strength and structural design that is critical for cancer cell motility, epithelial to mesenchymal transition (EMT), metastasis, and invasion. Ingenuity Pathway Analysis of highly expressed circulating proteins identified key oncogenic upstream transcriptional regulators and proteins that promote EMT, including KRAS, MYC and TGFB. Additionally, pathways involved in remodeling of epithelial adherens junctions, actin cytoskeleton signaling and VEGF signaling that also play a key role in EMT were upregulated. Additional datasets from proteomic profiling of multiple (N=6) GC cell lines were intersected with GC patient plasma profiles provided supporting evidence of a cancer cell origin of circulating cytoskeletal features. ACTC1, ACTN1, ACTR3, MYH9, TUBA1A, TUBA4A and TLN1 were highly expressed in both conditioned media released from GC cell lines and plasma samples of GC patients. Our findings are indicative of a blood-based signature of cytoskeletal proteins that is reflective of EMT in the context of gastric cancer. Citation Format: Ali H. Abdel Sater, Yihui Chen, Johannes F. Fahrmann, Ehsan Irajizad, Yining Cai, Fuchung Hsiao, Jody V. Vykoukal, Hiroyuki Katayama, Ricardo A. Léon-Letelier, Rongzhang Dou, Edwin J. Ostrin, Melissa Pizzi, Kohei Yamashita, Matheus Sewastjanow Da Silva, Shumei Song, Jaffer Ajani, Samir Hanash. A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2017.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase.

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.

Role of cytoskeleton-related proteins in the acrosome reaction of Eriocheir sinensis spermatozoa

Cytoskeleton-related proteins are essential for cell shape maintenance and cytoskeleton remodeling. The spermatozoa of Eriocheir sinensis (Chinese mitten crab) have a unique cellular structure, and the mechanism of spermatozoal metamorphosis during the acrosome reaction is not well understood. In this study, the E. sinensis spermatozoa were induced using calcium ionophore A23187 to undergo the acrosome reaction in vitro, and the acrosome-reacting and fresh (non-reacting) spermatozoa were collected separately. The differential expression of cytoskeleton-related protein genes in acrosome-reacting and fresh spermatozoa of E. sinensis was analyzed by whole transcriptome sequencing and bioinformatics analysis, and PPI network and miRNA-mRNA regulation network were constructed to analyze their possible function and regulation mechanism. The results showed that numerous differentially expressed cytoskeleton-related protein genes, miRNAs and lncRNAs were found in acrosome-reacting and fresh spermatozoa of E. sinensis; 27 cytoskeleton-related protein genes were down regulated and 687 miRNAs were up regulated in acrosome-reacting spermatozoa; 147 miRNAs target these 27 cytoskeleton-related protein genes. In the PPI networks, RAC1, BCAR1, RDX, NCKAP1, EPS8, CDC42BPA, LIMK1, ELMO2, GNAI1 and OCRL were identified as hub proteins. These proteins are mainly involved in the regulation of cytoskeleton organization, actin cytoskeleton organization, microtubule skeleton organization and small GTPase-mediated signal transduction and other biological processes, and play roles in pathways such as actin cytoskeletal regulation and axon guidance. miR-9, miR-31 and two novel miRNAs in the miRNA-mRNA regulatory network are the core miRNAs targeting cytoskeleton-related protein genes. miR-9 targets and regulates OBSCN, CDC42BPA, ELMO2, BCAS3, TPR and OCRL; while miR-31 targets and regulates CDC42BPA and TPR. This study provides a theoretical basis for revealing the mechanism of acrosome reaction under the special spermatozoa morphology of E. sinensis.

Transcriptome Analysis of Multiple Tissues in the Shrimp Penaeus vannamei Reveals the Typical Physiological Response to Three Pathogens

Penaeid shrimp aquaculture is impacted by various diseases. However, most published studies on physiological responses to pathogens have focused on the changes in one or two tissues of shrimp infected by a single pathogen, or the effects of two pathogens infecting the shrimp in a single tissue. There has been limited systematic examination on the similarities and differences of immune responses in multiple tissues under various pathogen infection. Here, the transcriptomic changes of three immune tissues (gill, hepatopancreas and hemocytes) under the infection of white spot syndrome virus (WSSV), Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND), and decapod iridovirus 1 (DIV1) were examined to provide new insights regarding the immune responses of the most important cultured shrimp, Penaeus vannamei. The results showed tissue-specific differences in the immune responses of shrimp tissues. The significant differentially expressed genes (DEGs) in gill are mainly related to environmental information processing and cellular processes. The DEGs in hemocytes are mostly involved in cellular processes, while those in hepatopancreas are primarily associated with metabolism. In addition, cytoskeleton-related proteins, MAPK signaling pathway, complement and coagulation level pathway, and thermogenesis may play key roles in the shrimp–pathogen interactions across tissues. These findings shed light on the typical immune responses of Penaeus vannamei under the infection of pathogens and contribute to the sustainable development of penaeid shrimp farming.

Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Abstract A016: Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility

Abstract Age is the major risk factor in most carcinomas, yet, little is known about the specific reasons aging increases cancer susceptibility. In the mammary gland, luminal epithelial cells rank high as the putative breast cancer cell of origin. Dysregulation of keratin intermediate filament proteins exemplifies a hallmark age-dependent change in luminal cells, which implicates mechanical states unique to cancer susceptible cells. We implemented mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell analysis that simultaneously measures cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We demonstrated that the epithelial lineages, chronological ages, and stages of cancer progression of primary human mammary epithelial cells (HMEC) exhibited discrete mechanical phenotypes. We trained a machine learning model that accurately predicted the chronological age of average risk HMEC cells based exclusively on mechanical properties. Application of the model to cells from women who are germline carriers of high-risk cancer-causing mutations showed that they are mechanically old irrespective of their chronological age, suggesting that mechanical states could be a window into detection and prevention of cancer susceptible states. Indeed, this mechano-age model detected high-risk women with &amp;gt;90% accuracy. Mass spectrometry and cell-based functional assays in mammary epithelia revealed that cytoskeleton related proteins keratin 14 (KRT14) and pseudopodium enriched atypical kinase 1 (PEAK1) were key drivers of age-dependent mechanical signatures. Pharmacological and gene silencing approaches that targeted KRT14 and PEAK1 modulated the mechanical age of HMEC and, in the case of PEAK1 modulation, ablated luminal epithelial cells in an age- and lineage- dependent manner. We define an intersection between mechanical phenotypes and novel age-dependent changes in cytoskeleton-related proteins that we hypothesize can be exploited to assess an individual’s breast cancer susceptibility and provide new targets for cancer-prevention strategies. Citation Format: Stefan Hinz, Masaru Miyano, Antigoni Manousopoulou, Rosalyn W. Sayaman, Kristina Y. Aguilera, Michael E. Todhunter, Jennifer C. Lopez, Leo D. Wang, Lydia L. Sohn, Mark A. LaBarge. Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A016.

Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood–brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

Proteomic Analysis Reveals Molecular Differences in the Development of Gastric Cancer.

Gastric cancer (GC) is the 3rd leading cause of death from cancer and the 5th most common cancer worldwide. The detection rate of GC among Tibetans is significantly higher than that in Han Chinese, probably due to differences in their living habits, dietary structure, and environment. Despite such a high disease burden, the epidemiology of gastric cancer has not been studied in this population. Molecular markers are required to aid the diagnosis and treatment of GC. In this study, we collected gastric tissue samples from patients in Tibet with chronic nonatrophic gastritis (CNAG) (n = 6), chronic atrophic gastritis (CAG) (n = 7), gastric intraepithelial neoplasia (GIN) (n = 4), and GC (n = 5). The proteins in each group were analyzed using coupled label-free mass spectrometry. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein interaction networks were used to analyze the differentially expressed proteins (DEPs) among groups. DEPs were quantified in comparisons of GC versus CNAG (223), GC versus GIN (100), and GIN versus CNAG (341). GO and KEGG analyses showed that the DEPs were mainly associated with immunity (GC versus CNAG) and cancer proliferation and metastasis (GC versus GIN, and GIN versus CNAG). Furthermore, the expression levels of cell proliferation and cytoskeleton-related proteins increased consistently during cancer development, such as ITGA4, DDC, and CPT1A; thus, they are potential diagnostic markers. These results obtained by proteomics analysis could improve our understanding of cancer biology in GC and provide a rich resource for data mining and discovering potential immunotherapy targets.