Ticks are notable vectors of diseases affecting both humans and animals, with Hyalomma anatolicum being of particular significance due to its wide distribution and capability to transmit a variety of pathogens, including Theileria annulata, and Crimean-Congo haemorrhagic fever (CCHF) virus. This study aimed to investigate the inhibitory effects of Hyalomma anatolicum salivary gland extract (HaSGE) and the identification of its key component on the complement system of the host\'s innate immune defense. We demonstrated that HaSGE exerts a dose-dependent inhibition on the complement activation in a host-specific manner. Mechanistic studies revealed that HaSGE interferes with blocking the deposition and cleavage of complement proteins C3 and C5, thus preventing the formation of the membrane attack complex (MAC). Further, we identified a serine protease inhibitor, HAMpin-1, from the HaSGE through proteomic analysis and characterized its structure, function, and interaction with complement proteins. HAMpin-1 exhibited potent inhibitory activity against chymotrypsin and cathepsin-G, and notably, it is the first serpin from ticks shown to inhibit the classical and lectin pathways of the complement system. The expression of HAMpin-1 was highest in the salivary glands, suggesting its crucial role in blood feeding and immune evasion. Our findings revealed one of the potential mechanisms employed by H. anatolicum to modulate host immune responses at the interface, offering new insights into tick-host interactions.

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.

Plasma membrane proteins (PMPs) play critical roles in a myriad of physiological and disease conditions. A unique subset of PMPs functions through interacting with each other in trans at the interface between two contacting cells. These trans-interacting PMPs (tiPMPs) include adhesion molecules and ligands/receptors that facilitate cell-cell contact and direct communication between cells. Among the tiPMPs, a significant number have apparent extracellular binding domains but remain orphans with no known binding partners. Identification of their potential binding partners is therefore important for the understanding of processes such as organismal development and immune cell activation. While a number of methods have been developed for the identification of protein binding partners in general, very few are applicable to tiPMPs, which interact in a two-dimensional fashion with low intrinsic binding affinities. In this review, we present the significance of tiPMP interactions, the challenges of identifying binding partners for tiPMPs, and the landscape of method development. We describe current avidity-based screening approaches for identifying novel tiPMP binding partners and discuss their advantages and limitations. We conclude by highlighting the importance of developing novel methods of identifying new tiPMP interactions for deciphering the complex protein interactome and developing targeted therapeutics for diseases.

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that can form at guanine-rich sequences of DNA and RNA in every kingdom of life. At the DNA level, G4s can form throughout genomes but they are prevalently found in promoter regions and at telomeres, and they have been attributed functions spanning from transcriptional regulation, to control of DNA replication, to maintenance of chromosome ends. Our understanding of the functions of G4s in cells has greatly improved with the development of specific anti-G4 antibodies, which allow the visualization of G4s by immunofluorescence but also the mapping of these secondary DNA structures genome wide. Whole genome identification of the location and abundance of G4s with techniques such as Chromatin Immunoprecipitation coupled with sequencing (ChIP-Seq) and Cleavage Under Target and Tagmentation (CUT&Tag) has allowed the profiling of G4 distribution across distinct cell types and deepen the understanding of G4 functions, particularly in the regulation of transcription. Crucial for these types of genome-wide studies is the availability of an anti-G4 antibody preparation with high affinity and specificity. Here, we describe a protocol for the expression and purification of the anti-DNA G4 structure antibody (BG4) first developed by the Balasubramanian group, which has been proven to selectively recognize G4 structures both in vitro and within cells, and which has great applicability in high-throughput techniques. We provide a detailed, step-by-step protocol to obtain active BG4 starting from a commercially available expression plasmid. We also describe three different approaches to validate the activity of the BG4 preparation.

RNA-binding proteins (RBPs) are at the heart of many biological processes and are therefore essential for cellular life. Following identification of single RBPs by classical genetics and molecular biology methods, approaches for RBP discovery on a systems level have recently emerged. For instance, RNA interactome capture (RIC) enables the global purification of RBPs cross-linked to polyadenylated RNA using oligo(dT) probes. RIC was originally developed for eukaryotic organisms but was recently established for capturing RBPs in bacteria. In this chapter, we provide a detailed step-by-step protocol for performing RIC in bacteria. The protocol is based on its application to Escherichia coli but should be amenable for charting other genetically tractable bacterial species.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

The development of higher-throughput, potentially lower-cost means to isolate proteins, for a variety of end uses, is of continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin-binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min-1 (295.2 cm h-1) with a SAV feed concentration of 0.5 mg mL-1 produces a SAV loading capacity of 1.4 mg g-1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10-14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25-0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays.

BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation.
METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells.
RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction.
CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.

Pull-down assay is a technique to analyze direct protein-protein interaction under in vitro condition. Also, this technique is appropriate for investigating the direct interaction between two purified proteins. Glutathione-s-transferase (GST) protein is a widely used affinity tag for affinity purification. In this chapter, we explain the widely used GST pull-down assay to identify the protein-protein interaction between purified proteins.

The glomerulus is the filtration unit of the kidney, where the first step of urine formation occurs. Podocytes are characterized by their actin-based projections called foot processes. Podocyte foot processes play a critical role in the permselective filtration barrier, along with fenestrated endothelial cells and the glomerular basement membrane. The Rho family of small GTPases (Rho GTPases) is the master regulators of the actin cytoskeleton and functions as molecular switches. Recent studies have shown that disruption of Rho GTPase activity and subsequent changes in foot process structure cause proteinuria. Here, we describe an effector pull-down assay using GST-fusion proteins to monitor the activity of RhoA, Rac1, and Cdc42, which are prototypical Rho GTPases in podocytes.