Interacting Proteins Research Articles

Abstract P2-11-33: Striatin and its interactive proteins Protein Phosphatase-2 (PP2) and PP2 regulatory elements in breast cancer

Abstract Introduction. The striatin protein family are calmodulin binding scaffolding proteins. Striatins have been involved in cell signalling and recently found also acting as cell adhesion molecules and adhesion regulators in adherens junctions and tight junctions. Straitins, particularly striatin-2 forms a recently discovered protein complex, STRIPAK, with other complex members including Protein Phosphatase-2 (PP2), STRIP, SIKE1 and certain PP2 regulatory proteins. The STRIPAK regulates multiple cell functions including gene transcription, endo- and exocytosis and cell adhesion, and several other important cell functions in cancer development and progression. The constitute members of the striatin protein complex may thus have important clinical bearings. In the present study, we investigated the expression profile of the striatin family members and key members of their interacting proteins, and discerned the relationship between the expression and disease progression and clinical outcome. Methods. Using an established fresh frozen breast cancer tissues cohort that included both normal mammary tissues and cancer tissues, we quantitatively evaluated the transcript expression of striatin-1 (STRN), striatin-3 (STRN3), straitin-4 (STRN4), its key regulator calmodulin (CALM) and the protein complex regulators protein phosphatase-2A (PPP2A), -2B (PPP2B) and the PPP2 regulatory elements PPP2R4 and PPP2R1A. The expression of each molecule was assessed against the clinical, pathological and prognostic factors of the patients. The integrated pattern of the complex members were also tested against the clinical outcome. Results. All three striatin members were expressed at good levels in mammary tissues and cancer tissues. STRN had little significant value against clinical and pathological factors. STRN3 and STRN4 were seen at high levels in tumours of high grade, with node positivity and with breast cancer related incidence. It was high level expressions of STRN3, STRN4 and CALM that were respectively associated with shorter overall survival (OS) of the patients and together they formed a poor prognostic indicator (p=0.034, HR=1.7). STRN had little impact on clinical outcomes. In a clear contrast, high levels of PPP2A, PPP2B and PPPR1A, but not PPP2R4, were seen in patients with significantly longer OS and together form a favourable prognostic indicator (p=0.034, HR=0.685). Integration of both STRN group and PPP2 group indicators constitutes a highly significant prognostic indicator for OS (p< 0.00001, HR=2.1 (95%CI 1.36-3.07)) and DFS (p=0.003, HR=1.402 (95%CI 1.12-1.75)). The predictive value of the integrated profile is independent of other clinical, pathological and hormone receptor status in multivariate analyses with OS (p< 0.0001, HR=3.861) and for DFS (p< 0.001, HR=2.055 (95%CI 1.36-3.07)). The same value stands when applied for the subtypes including triple negative breast cancers. Discussion. The STRIPAK complexes including Striatins play important regulatory roles in various cell functions and cancer development. Consistently, we found that high level expressions of STRN3, STRN4 and CALM, as a poor prognostic indicator were associated with shorter overall survival (OS) of the patients, while high levels of PPP2A, PPP2B and PPPR1A, as a favourable prognostic indicator, were seen in patients with significantly longer OS. Combination of both STRN group and PPP2 group indicators constitutes a greater significant prognostic indicator for OS and DFS. Future studies should focus on investigating the exact roles of the STRIPAK in cancer development and progression. Citation Format: Amber Xinyu Li, Andrew J. Sanders, Tracey A. Martin, Lin Ye, Fiona Ruge, QingPing Dou, Eleri Davies, Wen G. Jiang. Striatin and its interactive proteins Protein Phosphatase-2 (PP2) and PP2 regulatory elements in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-33.

Abstract P3-05-42: Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis

Abstract Introduction. The CUB domain containing protein 1, also known as TRASK (Transmembrane and associated with Src kinases) or CD318, has been suggested as a biomarker for stem cells or stem like cells. The transmembrane CDCP1 protein is known to activate the Src kinase and has recently shown to play a key part in the aggressiveness of breast cancer cells including migration, invasiveness and possibly growth induced by the motogenic Hepatocyte Growth Factor (HGF) and its receptor MET (Kawase et al 2022), a process requires Rho-GEF, PI3K and STAT5. This appears to allow identification of a subset of cancer cells that are aggressive in their biological behaviours. Here, we examined the expression profile of CDCP1, SRC, the HGF/HGF receptor complex and HGF activation regulators in a cohort of breast cancer and attempt to establish if such protein axis is able to identify patients with high risk of poor clinical outcome. Methods. Using an establish breast cancer cohort which is comprised of both normal mammary tissues and breast cancer tissues freshly obtained after surgery, we quantified the gene transcripts of CDCP1/CD318 and examined its clinical and pathological relevance. Integrated analysis was conducted for CDCP1 and the expression profile of SRC, PI3K, HGF, the HGF receptor cMET, HGF activator (HGFA), HGF activation regulators including HAI-1, HAI2, matriptase-1 and matriptase-2, and STAT family members. This was done against the clinical outcome of the patients as well as the clinical and pathological factors. Results. Breast cancer tissues expressed high levels of CDCP1 compared with normal mammary tissues. CDCP1 itself had a weak yet significant value in predicting the overall survival of the patients (p=0.047). The expression levels of these CDCP1 related molecules aren’t significantly correlated with CDCP1. However, integrated analyses revealed that CDCP1, together with its pathway regulators SRC, HGF, the HGF receptor (MET) and the HGF activation regulators matriptases form a power prognostic indicator for the clinical outcome of the patients (p< 0.0001, HR=1.8 for overall survival (OS) and p=0.002, HR=1.5 for disease-free survival (DFS)). This integrated profile has also identified subgroups of patients with highly favourable and very poor clinical outcomes over a ten-year follow-up period, with the respective survival at 100% and 36% respectively. The predictive power for OS and DFS is highly independent of other clinical and pathological factors in a multivariate analysis (p=0.003 and p=0.017 respectively). The predictive power is applicable to ER negative and HER2 positive tumours. Discussion. CDCP1/CD318, a factor known to stimulate cancer cell aggressiveness, together with its pathway kinase SRC and newly identified extracellular activator HGF and the HGF regulators forms a significant independent prognostic factor. It identifies subgroups of patients with favourable and poor prognosis, allowing consideration of targeted therapies for the patients. Reference Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S, Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells. J Biol Chem, 2022, 298:101630. doi: 10.1016/j.jbc.2022.101630 Citation Format: Yiming Yang, Xuefei Dong, Tracey A. Martin, Lin Ye, Jane Lane, Andrew J. Sanders, QingPing Dou, Eleri Davies, Wen G. Jiang. Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-42.

Abstract P2-26-20: Marvel D3 and its associated junctional proteins in breast cancer

Abstract Marvel D3 and its associated junctional proteins in breast cancer Wenxiao Ji, Wen G. Jiang, Tracey A. Martin CCMRC, Cardiff University School of Medicine, Cardiff, Wales, UK Introduction. Marvel (Membrane Associated Domain Containing) proteins are a small family of proteins that are concentrated at the tight junction (TJ) region of epithelial and endothelial cells. They are important players in the formation and regulation of TJs. The Marvel protein family has three members, Marvel-D1, Marvel-D2 and Marvel-D3, all known to be TJ components. Marvel-D3 is more prominent in TJ regulation, by interacting widely with other proteins that co-localised at TJs. In the present study, we have examined the expression of Marvel-D3 and its splicing variants, their relationship with other known interactive TJ partners in breast cancer and in disease progression and clinical outcome. Methods. Marvel-D3 and its variants were quantified in a breast cancer cohort that contained both normal and tumour tissues. The expression was analysed together with the known Marvel-D3 interactive molecules available in our database of the same cohort. The profile of the expression of the molecules was also integrated to search for a pattern of the expression that may have clinical significance including the survival of the patients. Results. Marvel-D3 had significantly high levels in breast cancer tissues than normal tissues (p=0.011). The difference between normal and tumour tissues for two Marvel-D3 variants were not statistically significant. Of all the known Marvel-D3 interactive proteins that were available for analyses, we identified thirteen with viability in integrated analyses; they include the JAM family members, Marvel-D2, occludin, the zonula occluden family members, small number of the claudin family members and cingulin, all being important TJ components. The integrated expression of these Marvel-D3 partners presented a highly significant predictive power for the overall survival of the patients (RUC=0.821, p< 0.0001). Stratifying the patients based on the profile has a significant value in evaluating survival (survival rate 95.7% with favourable expression versus 57.1% unfavourable, during the followup period). This predictive power is independent of other clinical and hormonal receptor status (p< 0.001, HR=1.226 (95% CI 1.094-1.373)). We have also noted that this predictive value is applicable to subtypes of the breast cancer and hormone receptor status with similar predictive power. Conclusion. Expression of Marvel-D3, a TJ component, together with its interactive TJ partners, including both transmembrane and intracellular subcoat proteins, form an important clinical indicator for clinical progression of breast cancer. Citation Format: WENXIAO JI, Wen G. Jiang, Tracey A. Martin. Marvel D3 and its associated junctional proteins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-20.

Ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of serum metabolomic characteristics in people with different vitamin D levels.

Vitamin D is a fat-soluble vitamin with multiple functions. However, the metabolism of people with different vitamin D concentrations is still unclear. Herein, we collected clinical data and analysed the serum metabolome of people with 25-hydroxyvitamin D (25[OH]D) ≥40 ng/mL (A), 30 ng/mL ≤25(OH)D <40 ng/mL (B) and 25(OH)D <30 ng/mL (C) by the ultra-high-performance liquid chromatography-tandem mass spectrometry method. We found that haemoglobin A1c, fasting blood glucose, fasting insulin, homeostasis model assessment of insulin resistance and thioredoxin interaction protein were enhanced, while HOMA-β was reduced with the decrease of 25(OH)D concentration. In addition, people in the C group were diagnosed with prediabetes or diabetes. Metabolomics analysis showed that seven, thirty-four and nine differential metabolites were identified in the groups B vs A, C vs A and C vs B, respectively. Metabolites associated with cholesterol metabolism and bile acid biosynthesis, such as 7-ketolithocholic acid, 12-ketolithocholic acid, apocholic acid, N-arachidene glycine and d-mannose 6-phosphate, were significantly upregulated in the C group compared with the A or B groups. In conclusion, the disorder of vitamin D metabolism may be related to cholesterol metabolism and bile acid biosynthesis. This study provided a basis for exploring the possible mechanism leading to abnormal vitamin D metabolism.

Network expansion of genetic associations defines a pleiotropy map of human cell biology

Interacting proteins tend to have similar functions, influencing the same organismal traits. Interaction networks can be used to expand the list of candidate trait-associated genes from genome-wide association studies. Here, we performed network-based expansion of trait-associated genes for 1,002 human traits showing that this recovers known disease genes or drug targets. The similarity of network expansion scores identifies groups of traits likely to share an underlying genetic and biological process. We identified 73 pleiotropic gene modules linked to multiple traits, enriched in genes involved in processes such as protein ubiquitination and RNA processing. In contrast to gene deletion studies, pleiotropy as defined here captures specifically multicellular-related processes. We show examples of modules linked to human diseases enriched in genes with known pathogenic variants that can be used to map targets of approved drugs for repurposing. Finally, we illustrate the use of network expansion scores to study genes at inflammatory bowel disease genome-wide association study loci, and implicate inflammatory bowel disease-relevant genes with strong functional and genetic support.

The Na+/H+ antiporter GbSOS1 interacts with SIP5 and regulates salt tolerance in Gossypium barbadense

Cotton is a globally cultivated economic crop and is a major source of natural fiber and edible oil. However, cotton production is severely affected by salt stress. Although Salt Overly Sensitive 1 (SOS1) is a well-studied Na+/H+ antiporter in multiple plant species, little is known about its function and regulatory mechanism in cotton. Here, we cloned a salt-induced SOS1 from sea-island cotton. Real-time quantitative PCR analysis revealed that GbSOS1 was induced by multiple stresses and phytohormones. Silencing GbSOS1 through virus-induced gene silencing significantly reduced cotton resistance to high Na+ but mildly affected Li+ tolerance. On the other hand, overexpression of GbSOS1 enhanced salt tolerance in yeast, Arabidopsis, and cotton largely due to the ability to maintain Na+ homeostasis in protoplasts. Yeast-two-hybrid assays and bimolecular fluorescence complementation identified a novel protein interacting with GbSOS1 on the plasma membrane, which we named SOS Interaction Protein 5 (SIP5). We found that the SIP5 gene encoded an unknown protein localized on the cell membrane. Silencing SIP5 significantly increased cotton tolerance to salt, exhibited by less wilting and plant death under salt stress. Our results revealed that GbSOS1 is crucial for cotton survival in saline soil, and SIP5 is a potentially negative regulator of SOS1-mediated salt tolerance in cotton. Overall, this study provides a theoretical basis for elucidating the molecular mechanism of SOS1, and a candidate gene for breeding salt-tolerant crops.

SmCIP7, a COP1 interactive protein, positively regulates anthocyanin accumulation and fruit size in eggplant

In higher plants, COP1 (Constitutively Photomorphogenic 1) acts as a central regulator of light-signaling networks and globally conditions the target proteins via the ubiquitin-proteasome pathway. However, the function of COP1-interacting proteins in light-regulated fruit coloration and development remains unknown in Solanaceous plants. Here, a COP1-interacting protein-encoding gene, SmCIP7, expressed specifically in the eggplant (Solanum melongena L.) fruit, was isolated. Gene-specific silencing of SmCIP7 using RNA interference (RNAi) significantly altered fruit coloration, fruit size, flesh browning, and seed yield. SmCIP7-RNAi fruits showed evident repression of the accumulation of anthocyanins and chlorophyll, indicating functional similarities between SmCIP7 and AtCIP7. However, the reduced fruit size and seed yield indicated SmCIP7 had evolved a distinctly new function. With the comprehensive application of HPLC-MS, RNA-seq, qRT-PCR, Y2H, BiFC, LCI, and dual-luciferase reporter system (DLR™), it was found that SmCIP7, a COP1 interactive protein in light signaling promoted anthocyanin accumulation, probably by regulating the transcription of SmTT8. Additionally, the drastic up-regulation of SmYABBY1, a homologous gene of SlFAS, might account for the strongly retarded fruit growth in SmCIP7-RNAi eggplant. Altogether, this study proved that SmCIP7 is an essential regulatory gene to modulate fruit coloration and development, serving as a key gene locus in eggplant molecular breeding.

Genome-centric view of the microbiome in a new deep-sea glass sponge species Bathydorus sp.

Sponges are widely distributed in the global ocean and harbor diverse symbiotic microbes with mutualistic relationships. However, sponge symbionts in the deep sea remain poorly studied at the genome level. Here, we report a new glass sponge species of the genus Bathydorus and provide a genome-centric view of its microbiome. We obtained 14 high-quality prokaryotic metagenome-assembled genomes (MAGs) affiliated with the phyla Nitrososphaerota, Pseudomonadota, Nitrospirota, Bdellovibrionota, SAR324, Bacteroidota, and Patescibacteria. In total, 13 of these MAGs probably represent new species, suggesting the high novelty of the deep-sea glass sponge microbiome. An ammonia-oxidizing Nitrososphaerota MAG B01, which accounted for up to 70% of the metagenome reads, dominated the sponge microbiomes. The B01 genome had a highly complex CRISPR array, which likely represents an advantageous evolution toward a symbiotic lifestyle and forceful ability to defend against phages. A sulfur-oxidizing Gammaproteobacteria species was the second most dominant symbiont, and a nitrite-oxidizing Nitrospirota species could also be detected, but with lower relative abundance. Bdellovibrio species represented by two MAGs, B11 and B12, were first reported as potential predatory symbionts in deep-sea glass sponges and have undergone dramatic genome reduction. Comprehensive functional analysis indicated that most of the sponge symbionts encoded CRISPR-Cas systems and eukaryotic-like proteins for symbiotic interactions with the host. Metabolic reconstruction further illustrated their essential roles in carbon, nitrogen, and sulfur cycles. In addition, diverse putative phages were identified from the sponge metagenomes. Our study expands the knowledge of microbial diversity, evolutionary adaption, and metabolic complementarity in deep-sea glass sponges.

5 - Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

5 - Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Differential Urinary Proteomic Analysis of High-Risk Cervical Intraepithelial Neoplasia.

Human papillomavirus (HPV)-associated lesions and malignancies exhibit alterations in the composition and functionality of the extracellular matrix (ECM) that represent the complex molecular pathways present between infection and disease. A total of 20 urine samples were used, including from 10 patients with cervical intraepithelial neoplasia grade 3 (CIN3) and 10 healthy controls to perform the label-free quantitative analysis using the nano-HPLC and ESI-MS ion trap mass analyzer and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) fast screening. Among 476 identified/quantified proteins, 48 were significantly changed (log2-fold change ≥1.0 or ≤-1.0, -log10 (bbinominal, p-value ≥ 1.3), of which were 40 proteins (down-regulated) and 8 proteins (up-regulated) in CIN3, in comparison to healthy controls. The biological function and key pathway enrichment of the gene set using gen set enrichment analysis (GSEA) were analyzed. The ECM-receptor interaction pathway (NES = -1.64, p = 0.026) was down-regulated by 13 proteins (HSPG2, COL6A1, COL6A3, SPP1, THBS1, TNC, DAG1, FN1, COMP, GP6, VTN, SDC1, and CD44; log2 FC range from -0.03 to -1.48) for the CIN3 group in the KEGG database. The MALDI-TOF/MS screening showed the difference of protein profiles between the control and CIN3 groups, i.e., using the scatter plot with a well-separated shape, as well as effectively distinguishing both groups (control and CIN3) using genetic algorithms (GA) with cross-validation (51.56%) and recognition capability (95.0%). Decreased levels of ECM-receptor interaction proteins may cause disturbances in the interactions of cells with the ECM and play an important role in the development and progression of cervical cancer.

H+-pyrophosphatases enhance low nitrogen stress tolerance in transgenic Arabidopsis and wheat by interacting with a receptor-like protein kinase.

Nitrogen is a major abiotic stress that affects plant productivity. Previous studies have shown that plant H+-pyrophosphatases (H+-PPases) enhance plant resistance to low nitrogen stress. However, the molecular mechanism underlying H+-PPase-mediated regulation of plant responses to low nitrogen stress is still unknown. In this study, we aimed to investigate the regulatory mechanism of AtAVP1 in response to low nitrogen stress. AtAVP1 in Arabidopsis thaliana and EdVP1 in Elymus dahuricus belong to the H+-PPase gene family. In this study, we found that AtAVP1 overexpression was more tolerant to low nitrogen stress than was wild type (WT), whereas the avp1-1 mutant was less tolerant to low nitrogen stress than WT. Plant height, root length, aboveground fresh and dry weights, and underground fresh and dry weights of EdVP1 overexpression wheat were considerably higher than those of SHI366 under low nitrogen treatment during the seedling stage. Two consecutive years of low nitrogen tolerance experiments in the field showed that grain yield and number of grains per spike of EdVP1 overexpression wheat were increased compared to those in SHI366, which indicated that EdVP1 conferred low nitrogen stress tolerance in the field. Furthermore, we screened interaction proteins in Arabidopsis; subcellular localization analysis demonstrated that AtAVP1 and Arabidopsis thaliana receptor-like protein kinase (AtRLK) were located on the plasma membrane. Yeast two-hybrid and luciferase complementary imaging assays showed that the AtRLK interacted with AtAVP1. Under low nitrogen stress, the Arabidopsis mutants rlk and avp1-1 had the same phenotypes. These results indicate that AtAVP1 regulates low nitrogen stress responses by interacting with AtRLK, which provides a novel insight into the regulatory pathway related to H+-pyrophosphatase function in plants.

ANXA2 as a novel substrate of FBXW7 promoting esophageal squamous cell carcinoma via ERK phosphorylation

Our recent study suggests that FBXW7 loss of function plays a critical function in esophageal cancer. However, the mechanism of FBXW7 in promoting esophageal cancer is still unclear. Here, we explored the interaction protein of FBXW7 by screening of GST-pulldown and LC-MS/MS analysis in esophageal squamous cell carcinoma (ESCC) and identified ANXA2 as a potential target of FBXW7. FBXW7 loss of function could restore the expression of ANXA2 and promote the malignant biological characteristics of ESCC cells in vitro. Up-regulation of ANXA2 enhances the ERK pathway in ESCC. Furthermore, the 23rd tyrosine residue of ANXA2, phosphorylated by SRC, was regarded as playing important roles in the FBXW7-related degradation system. In clinical samples, we found that ANXA2 had high expression in ESCC tissues. High ANXA2 was associated with poor tumor staging. More importantly, we designed a combination regimen including SCH779284, a clinical ERK inhibitor against the phosphorylation of EKR and siRNA targeting ANXA2 by intratumor injection, and it produced potent inhibitory effects on the growth of xenograft tumors in vivo. In conclusion, this study provided evidence that FBXW7 loss of function could promote esophageal cancer through ANXA2 overexpression, and this novel regulation pathway may be used as an efficient target for ESCC treatment.

Profiling Glycosylphosphatidylinositol (GPI)-Interacting Proteins in the Cell Membrane Using a Bifunctional GPI Analogue as the Probe.

Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.

Analysis of the role of BrRPP1 gene in Chinese cabbage infected by Plasmodiophora brassicae.

The clubroot disease caused by Plasmodiophora brassicae (P. brassicae) poses a serious threat to the economic value of cruciferous crops, which is a serious problem to be solved worldwide. Some resistance genes to clubroot disease in Brassica rapa L. ssp pekinensis cause by P. brassicae have been located on different chromosomes. Among them, Rcr1 and Rcr2 were mapped to the common candidate gene Bra019410, but its resistance mechanism is not clear yet. In this experiment, the differences of BrRPP1 between the resistant and susceptible material of Chinese cabbage were analyzed by gene cloning and qRT-PCR. The gene function was verified by Arabidopsis homologous mutants. The expression site of BrRPP1 gene in cells was analyzed by subcellular localization. Finally, the candidate interaction protein of BrRPP1 was screened by yeast two-hybrid library. The results showed that the cDNA sequence, upstream promoter sequence and expression level of BrRPP1 were quite different between the resistant and susceptible material. The resistance investigation found that the Arabidopsis mutant rpp1 was more susceptible to clubroot disease than the wild type, which suggested that the deletion of rpp1 reduces resistance of plant to clubroot disease. Subcellular location analysis confirmed that BrRPP1 was located in the nucleus. The interaction proteins of BrRPP1 screened from cDNA Yeast Library by yeast two-hybrid are mainly related to photosynthesis, cell wall modification, jasmonic acid signal transduction and programmed cell death. BrRPP1 gene contains TIR-NBS-LRR domain and belongs to R gene. The cDNA and promoter sequence of BrRPP1 in resistant varieties was different from that in susceptible varieties led to the significant difference of the gene expression of BrRPP1 between the resistant varieties and the susceptible varieties. The high expression of BrRPP1 gene in resistant varieties enhanced the resistance of Chinese cabbage to P. brassicae, and the interaction proteins of BrRPP1 are mainly related to photosynthesis, cell wall modification, jasmonic acid signal transduction and programmed cell death. These results provide important clues for understanding the mechanism of BrRPP1 in the resistance of B. rapa to P. brassicae.

Downregulation of ITGβ3 in colon adenocarcinoma reveals poor prognosis by affecting genome stability, cell cycle, and the tumor immune microenvironment.

Abnormal expression of integrin subunit beta 3 (ITGβ3), a gene-encoding protein, is related to the occurrence and development of cancers; however, the biological role of ITGβ3 in colon adenocarcinoma (COAD) remains unclear. We used the Cancer Genome Atlas database to obtain the clinical data of patients with COAD, analyzed the mRNA gene clusters related to ITGβ3, and analyzed the interaction signal pathway and interaction protein network of the differentially expressed gene clusters. The results showed that ITGβ3 expression in COAD tumor tissues was significantly downregulated compared with that in paracancerous tissues. Low ITGβ3 expression in tumor tissues is associated with poor overall survival of patients with COAD. In multivariate analysis, stage IV and ITGβ3 low expression were independent prognostic factors. Gene Ontology analysis showed that differentially expressed genes (DEGs) were significantly enriched in leukocyte migration, cell adhesion, and extracellular matrix (ECM) organization. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the DEGs were mainly enriched in ECM-receptor interactions, focal adhesion, and the PI3K-Akt signaling pathway. Protein-protein interaction network analysis revealed the hub and seed genes of the key modules related to ITGβ3. Finally, we analyzed the correlation between TGβ3 and immune-related genes and found that ITGβ3 expression was significantly correlated with tumor purity and infiltration level of dominant immune cells. These findings indicate that ITGβ3 downregulation in COAD may profoundly affect genome stability and multiple steps of the cell cycle, alter the tumor immune microenvironment, and be related to the prognosis of patients with COAD.

ACE2 PET to reveal the dynamic patterns of ACE2 recovery in an infection model with pseudocorona virus.

Due to the COVID-19 pandemic, a series of sequelae, such as fatigue, tachypnea, and ageusia, appeared in long COVID patients, but the pathological basis was still uncertain. The targeted radiopharmaceuticals were of potential to systemically and dynamically trace the pathological changes. For the key ACE2 protein in the virus-host interaction, 68 Ga-cyc-DX600 was developed on the basis of DX600 as a PET tracer of ACE2 fluctuation and maintained the ability in differentiating ACE and ACE2. In the temporary infection model inhaled with the radio-traceable pseudovirus in the upper respiratory tract of male humanized ACE2 (hACE2) mice, organ-specific ACE2 dysfunction in acute period and the following ACE2 recovery in a relatively long period was visualized and quantified by ACE2 PET, revealing a complex pattern of virus concentration-dependent degree and time period-dependent tendency of ACE2 recovery, mainly a sudden decrease of apparent ACE2 in the heart, liver, kidneys, lungs, and so on, but the liver was of a quick functional compensation on ACE2 expression after a temporary decrease. ACE2 expression of most organs has recovered to a normal level at 15 days post inhalation, with brain and genitals still of a decreased SUVACE2 ; meanwhile, kidneys were of an increased SUVACE2 . These findings on ACE2 PET were further verified by western blot. When compared with high-resolution computed tomography on structural changes and FDG PET on glycometabolism, ACE2 PET was superior in an earlier diagnostic window during infection and more comprehensive understanding of functional dysfunction post-infection. In the respective ACE2 PET/CT and ACE2 PET/MR scans of a volunteer, the repeatability of SUVACE2 and the ACE2 specificity were further confirmed. In conclusion, 68 Ga-cyc-DX600 was developed as an ACE2-specific tracer, and the corresponding ACE2 PET revealed the dynamic patterns of functional ACE2 recovery and provided a reference and approach to explore the ACE2-related pathological basis of sequelae in long COVID.

VaSUS2 confers cold tolerance in transgenic tomato and Arabidopsis by regulation of sucrose metabolism and ROS homeostasis.

VaSUS2 enhances cold tolerance of transgenic tomato and Arabidopsis by regulating sucrose metabolism and improving antioxidant enzymes activity. Sucrose synthetase (SUS) is a key enzyme of sugar metabolism, and plays an important role in response to abiotic stress in plant. However, the function of VaSUS2 remains unknown in cold tolerance. Here, the cloning and functional characterization of the plasma membrane-localized VaSUS2 gene isolated from Vitis amurensis was studied. The transcript level of VaSUS2 was up-regulated under cold stress in Vitis amurensis. Heterologous expression of VaSUS2 in tomato increased SUS activity, which promoted the accumulation of glucose and fructose under cold treatment. The transgenic tomato and Arabidopsis exhibited higher levels of antioxidant enzymes activity, lower relative electrolyte leakage (REL), malondialdehyde (MDA) and hydrogen peroxide (H2O2) content compared to wild type under cold stress. Importantly, the ability of scavenging reactive oxygen species (ROS) in transgenic plants was significantly improved. Moreover, yeast two-hybrid (Y2H) indicated that VaSnRK1 might be a potential interaction protein of VaSUS2. qRT-PCR showed that sucrose metabolism-related genes SlSUS, SlSPS and SlINV were significantly up-regulated in transgenic tomatoes. Meanwhile, the expression levels of antioxidant enzyme genes and cold-related genes CBF1, COR47 and ICE1 were up-regulated in transgenic plants. Taken together, these results suggested that VaSUS2 was involved in cold tolerance by increasing the levels of soluble sugars, improving the activity of antioxidant enzymes, and up-regulating the expression of cold-related genes in transgenic tomatoes and Arabidopsis.

In silico investigations of the multi‐targeted antiviral potential of small molecule phytochemicals of Nelumbo nucifera Gaertn. seed extracts against SARS‐CoV‐2 for therapeutics of COVID‐19

AbstractThe therapeutics of COVID‐2 was significantly obtained from nutraceutical approaches based on traditional knowledges and long practices. The current study was aimed to investigate antiviral potential of small molecule phytochemicals of seeds of Nelumbo nucifera against the SARS‐CoV‐2 proteins. GC–MS analysis resulted in identifying 12 dominating small molecule phytocompounds from the seed extracts of the N. nucifera. Further, we selected RNA‐dependent RNA polymerases (RdRps), spike protein, and M protein of SARS‐CoV‐2 as antiviral targets and performed in silico analyses, including protein docking (ligands), ADMET predictions, and molecular dynamics (MD). The studies revealed that the molecular interactions (protein–ligands) of three target proteins, namely, RdRp, spike protein, and M‐protein, have significant binding energies with three different substrates, namely, 1‐(8′‐methylquinolin‐2′‐yl)‐2,3,4‐tri(methoxycarbonyl)‐6‐(1″,2″‐di(methoxycarbonyl)vinyloxy)benzene (binding energy −5.84 kcal/mol) 2(1H)‐pyrimidinone, 5‐chloro‐4,6‐diphenyl (binding energy −6.60 kcal/mol), and nickel, [2,8,12,18‐tetraethyl‐3,7,13,17‐tetramethyl‐21H,23H‐porphinato(2‐)‐N21,N22,N23,N24]‐, (SP‐4‐1) (binding energy −7.02 kcal/mol), respectively. The ADMET predictions show significant pharmacokinetic profiles of the druggability of the three compounds of different targets. The pharmacokinetic activities critical to predicting stages of the drug development process are gastrointestinal absorption and brain access. The MD simulation showed that the systems were stable, referring to them as a potentially effective treatment for SARS‐CoV‐2. Therefore, a possible initiative has been taken to evaluate three potent small molecules of antiviral phytocompounds made from typically edible N. nucifera seed to support the nutraceutical approach to COVID‐19 therapeutics.

Evolutionary dissection of monkeypox virus: Positive Darwinian selection drives the adaptation of virus-host interaction proteins.

The emerging and ongoing outbreak of human monkeypox (hMPX) in 2022 is a serious global threat. An understanding of the evolution of the monkeypox virus (MPXV) at the single-gene level may provide clues for exploring the unique aspects of the current outbreak: rapidly expanding and sustained human-to-human transmission. For the current investigation, alleles of 156 MPXV coding genes (which account for >95% of the genomic sequence) have been gathered from roughly 1,500 isolates, including those responsible for the previous outbreaks. Using a range of molecular evolution approaches, we demonstrated that intra-species homologous recombination has a negligible effect on MPXV evolution. Despite the fact that the majority of the MPXV genes (64.10%) were subjected to negative selection at the whole gene level, 10 MPXV coding genes (MPXVgp004, 010, 012, 014, 044, 098, 138, 178, 188, and 191) were found to have a total of 15 codons or amino acid sites that are known to evolve under positive Darwinian selection. Except for MPXVgp138, almost all of these genes encode proteins that interact with the host. Of these, five ankyrin proteins (MPXVgp004, 010, 012, 178, and 188) and one Bcl-2-like protein (MPXVgp014) are involved in poxviruses' host range determination. We discovered that the majority (80%) of positive amino acid substitutions emerged several decades ago, indicating that these sites have been under constant selection pressure and that more adaptable alleles have been circulating in the natural reservoir. This finding was also supported by the minimum spanning networks of the gene alleles. The three positive amino acid substitutions (T/A426V in MPXVgp010, A423D in MPXVgp012, and S105L in MPXVgp191) appeared in 2019 or 2022, indicating that they would be crucial for the virus' eventual adaptation to humans. Protein modeling suggests that positive amino acid substitutions may affect protein functions in a variety of ways. Further study should focus on revealing the biological effects of positive amino acid substitutions in the genes for viral adaptation to humans, virulence, transmission, and so on. Our study advances knowledge of MPXV's adaptive mechanism and provides insights for exploring factors that are responsible for the unique aspects of the current outbreak.

Genome-wide identification of acyl-CoA binding proteins and possible functional prediction in legumes.

Acyl-CoA-binding proteins (ACBPs), members of a vital housekeeping protein family, are present in various animal and plant species. They are divided into four classes: small ACBPs (class I), ankyrin-repeat ACBPs (class II), large ACBPs (class III), and kelch-ACBPs (class IV). Plant ACBPs play a pivotal role in intracellular transport, protection, and pool formation of acyl-CoA esters, promoting plant development and stress response. Even though legume crops are important for vegetable oils, proteins, vegetables and green manure, legume ACBPs are not well investigated. To comprehensively explore the functions of ACBPs in nine legumes (Lotus japonicus, Medicago truncatula, Glycine max, Vigna angularis, Vigna radiata, Phaseolus vulgaris, Arachis hypogaea, Arachis duranensis, and Arachis ipaensis), we conducted genome-wide identification of the ACBP gene family. Our evolutionary analyses included phylogenetics, gene structure, the conserved motif, chromosomal distribution and homology, subcellular localization, cis-elements, and interacting proteins. The results revealed that ACBP Orthologs of nine legumes had a high identity in gene structure and conserved motif. However, subcellular localization, cis-acting elements, and interaction protein analyses revealed potentially different functions from previously reported. The predicted results were also partially verified in Arachis hypogaea. We believe that our findings will help researchers understand the roles of ACBPs in legumes and encourage them to conduct additional research.