Complex Of Protein Research Articles

Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture, preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

Construction of Homozygous Mutants of Migratory Locust using CRISPR/Cas9 Technology.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.

An Engineered Protein-Au Bioplaster for Efficient Skin Tumor Therapy.

Melanoma is the most lethal malignancy in skin cancer and may occur at any site and express melanocytes. Due to malignant melanoma's invasion and migration nature, conventional therapies make it challenging to remove the whole tumor tissue while undertaking the high risks of tumor recurrence. Regarding the emerging targeted therapies and immunotherapy, drug resistance and low immunotherapeutic activity remain significant challenges. It is thus becoming urgently important to develop alternative strategies for melanoma therapy. Herein, a novel bifunctional protein-based photothermal bioplaster (PPTB) is developed for non-invasive tumor therapy and skin tissue regeneration. The complexation of adhesive protein and gold nanorods (GNRs) endow the obtained PPTB with good biocompatibility, controllable near-infrared (NIR) light-mediated adhesion performance, and high photothermal performance. Therefore, the PPTB bioagent facilitates skin adhesion and effectively transfers heat from skin to tumor. This behavior endows PPTB capability to eradicate skin tumors conveniently. Thus, the assembly strategy enables this hybrid bioplaster to hold great potential for skin-related tumor treatment.

Venom profile of the European carpenter bee Xylocopa violacea: Evolutionary and applied considerations on its toxin components.

Modern venomics is increasing its focus on hymenopterans such as honeybees, bumblebees, parasitoid wasps, ants and true wasps. However solitary bees remain understudied in comparison and the few available venom studies focus on short melittin-like sequences and antimicrobial peptides. Herein we describe the first comprehensive venom profile of a solitary bee, the violet carpenter bee Xylocopa violacea, by using proteo-transcriptomics. We reveal a diverse and complex venom profile with 43 different protein families identified from dissected venom gland extracts of which 32 are also detected in the defensively injected venom. Melittin and apamin are the most highly secreted components, followed by Phospholipase A2, Icarapin, Secapin and three novel components. Other components, including eight novel protein families, are rather lowly expressed. We further identify multiple forms of apamin-like peptides. The melittin-like sequences of solitary bees separate into two clades, one comprised most sequences from solitary bees including xylopin (the variant in Xylocopa), while sequences from Lasioglossa appear closer related to melittin-like peptides from Bombus (Bombolittins). Our study suggests that more proteo-transcriptomic data from other solitary bees should be complemented with corresponding genome data to fully understand the evolution and complexity of bee venom proteins, and is of a particular need to disentangle the ambiguous phylogenetic relations of short peptides.

Ultrasound-induced red bean protein–lutein interactions and their effects on physicochemical properties, antioxidant activities and digestion behaviors of complexes

The complexation of red bean protein (RBP) and lutein (LU) enhances the functional properties of proteins and increases their applicability as nutraceuticals for LU. The effects of ultrasonic treatment on the interactions between RBP and LU, physicochemical properties, antioxidant activities, and digestion behaviors of RBP–LU complexes were investigated. Ultrasonic treatment promoted the non-covalent binding of RBP with LU through hydrophobic interactions, causing the binding site to be closer to tryptophan than tyrosine. This process resulted in a more flexible secondary structure and less compact tertiary conformation of complexes with higher binding ratios, surface hydrophobicity, surface charge, and smaller particle size. The enhanced RBP–LU interactions and structural modifications of complexes induced by ultrasound possibly enhanced the functional properties. Ultrasonic treatment maximized the emulsifying and antioxidant activities of these complexes, and also enhanced the release of LU in the intestine by altering the digestion behavior of the complex, thereby improving its bioaccessibility. An appropriate ultrasonic treatment could facilitate RBP–LU interactions and modify the structure of complexes to improve their functional properties.

Alternative Splicing and Isoforms: From Mechanisms to Diseases.

Alternative splicing of pre-mRNA is a key mechanism for increasing the complexity of proteins in humans, causing a diversity of expression of transcriptomes and proteomes in a tissue-specific manner. Alternative splicing is regulated by a variety of splicing factors. However, the changes and errors of splicing regulation caused by splicing factors are strongly related to many diseases, something which represents one of this study’s main interests. Further understanding of alternative splicing regulation mediated by cellular factors is also a prospective choice to develop specific drugs for targeting the dynamic RNA splicing process. In this review, we firstly concluded the basic principle of alternative splicing. Afterwards, we showed how splicing isoforms affect physiological activities through specific disease examples. Finally, the available treatment methods relative to adjusting splicing activities have been summarized.

EGFR signaling promotes nuclear translocation of plasma membrane protein TSPAN8 to enhance tumor progression via STAT3-mediated transcription.

TSPAN family of proteins are generally considered to assemble as multimeric complexes on the plasma membrane. Our previous work uncovered that TSPAN8 can translocate into the nucleus as a membrane-free form, a process that requires TSPAN8 palmitoylation and association with cholesterol to promote its extraction from the plasma membrane and subsequent binding with 14-3-3θ and importin-β. However, what upstream signal(s) regulate(s) the nuclear translocation of TSPAN8, the potential function of TSPAN8 in the nucleus, and the underlying molecular mechanisms all remain unclear. Here, we demonstrate that, epidermal growth factor receptor (EGFR) signaling induces TSPAN8 nuclear translocation by activating the kinase AKT, which in turn directly phosphorylates TSPAN8 at Ser129, an event essential for its binding with 14-3-3θ and importin ß1. In the nucleus, phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and therefore regulates transcription of downstream cancer-promoting genes, such as MYC, BCL2, MMP9, etc. The EGFR-AKT-TSPAN8-STAT3 axis was found to be hyperactivated in multiple human cancers, and associated with aggressive phenotype and dismal prognosis. We further developed a humanized monoclonal antibody hT8Ab4 that specifically recognizes the large extracellular loop of TSPAN8 (TSPAN8-LEL), thus being able to block the extraction of TSPAN8 from the plasma membrane and consequently its nuclear localization. Importantly, both in vitro and in vivo studies demonstrated an antitumor effect of hT8Ab4. Collectively, we discovered an unconventional function of TSPAN8 and dissected the underlying molecular mechanisms, which not only showcase a new layer of biological complexity of traditional membrane proteins, but also shed light on TSPAN8 as a novel therapeutic target for refractory cancers.

Disruption of Trip11 in cranial neural crest cells is associated with increased ER and Golgi stress contributing to skull defects in mice.

Absence of Golgi microtubule-associated protein 210 (GMAP210), encoded by the TRIP11 gene, results in achondrogenesis. Although TRIP11 is thought to be specifically required for chondrogenesis, human fetuses with the mutation of TRIP11 also display bony skull defects where chondrocytes are usually not present. This raises an important question of how TRIP11 functions in bony skull development. We disrupted Trip11 in neural crest-derived cell populations, which are critical for developing skull in mice. In Trip11 mutant skulls, expression levels of ER stress markers were increased compared to controls. Morphological analysis of electron microscopy data revealed swollen ER in Trip11 mutant skulls. Unexpectedly, we also found that Golgi stress increased in Trip11 mutant skulls, suggesting that both ER and Golgi stress-induced cell death may lead to osteopenia-like phenotypes in Trip11 mutant skulls. These data suggest that Trip11 plays pivotal roles in the regulation of ER and Golgi stress, which are critical for osteogenic cell survival. We have recently reported that the molecular complex of ciliary protein and GMAP210 is required for collagen trafficking. In this paper, we further characterized the important role of Trip11 being possibly involved in the regulation of ER and Golgi stress during skull development.

A small-molecule inhibitor of MDMX suppresses cervical cancer cells via the inhibition of E6-E6AP-p53 axis

Dysfunction of p53 is observed in many malignant tumors, which is related to cancer susceptibility. In cervical cancer, p53 is primarily degradated through the complex of high-risk human papillomaviruses (HPV) oncoprotein E6 and E6-associated protein (E6AP) ubiquitin ligase. What is less clear is the mechanism and role of murine double minute X (MDMX) in cervical carcinogenesis due to the inactive status of murine double minute 2 (MDM2). In the current study, XI-011 (NSC146109), a small-molecule inhibitor of MDMX, showed robust anti-proliferation activity against several cervical cancer cell lines. XI-011 promoted apoptosis of cervical cancer cells via stabilizing p53 and activating its transcription activity. Moreover, XI-011 inhibited the growth of xenograft tumor in HeLa tumor-bearing mice, as well as enhanced the cytotoxic activity of cisplatin both in vitro and in vivo. Interestingly, MDMX co-localized with E6AP and seems to be a novel binding partner of E6AP to promote p53 ubiquitination. In conclusion, this work revealed a novel mechanism of ubiquitin-dependent p53 degredation via MDMX-E6AP axis in cervical carcinogenesis, and offered the first evidence that MDMX could be a viable drug target for the treatment of cervical cancer.

Sequence-based prediction of protein binding regions and drug\u2013target interactions

Identifying drug–target interactions (DTIs) is important for drug discovery. However, searching all drug–target spaces poses a major bottleneck. Therefore, recently many deep learning models have been proposed to address this problem. However, the developers of these deep learning models have neglected interpretability in model construction, which is closely related to a model’s performance. We hypothesized that training a model to predict important regions on a protein sequence would increase DTI prediction performance and provide a more interpretable model. Consequently, we constructed a deep learning model, named Highlights on Target Sequences (HoTS), which predicts binding regions (BRs) between a protein sequence and a drug ligand, as well as DTIs between them. To train the model, we collected complexes of protein–ligand interactions and protein sequences of binding sites and pretrained the model to predict BRs for a given protein sequence–ligand pair via object detection employing transformers. After pretraining the BR prediction, we trained the model to predict DTIs from a compound token designed to assign attention to BRs. We confirmed that training the BRs prediction model indeed improved the DTI prediction performance. The proposed HoTS model showed good performance in BR prediction on independent test datasets even though it does not use 3D structure information in its prediction. Furthermore, the HoTS model achieved the best performance in DTI prediction on test datasets. Additional analysis confirmed the appropriate attention for BRs and the importance of transformers in BR and DTI prediction. The source code is available on GitHub (https://github.com/GIST-CSBL/HoTS).

Identifying and characterizing effectors of low complexity protein domain assembly

TDP-43 is an RNA and DNA processing protein with a highly conserved, C-terminal low-complexity domain. In vitro, the TDP-43 low complexity domain can form liquid-liquid phase separations which readily mature into amyloid fibrils. Fibrils containing C-terminal fragments of TDP-43 have been identified within cellular inclusions of post-mortem neuronal tissue in a considerable range of neurodegenerative diseases, including ALS, FTD, and Alzheimer’s. Nearly all ALS-associated mutations are found in the low complexity domain, further highlighting its significance to disease.

The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles

Histidine-rich peptides confer self-assembling properties to recombinant proteins through the supramolecular coordination with divalent cations. This fact allows the cost-effective, large-scale generation of microscopic and macroscopic protein materials with intriguing biomedical properties. Among such materials, resulting from the simple bioproduction of protein building blocks, homomeric nanoparticles are of special value as multivalent interactors and drug carriers. Interestingly, we have here identified that the assembly of a given His-tagged protein might render distinguishable categories of self-assembling protein nanoparticles. This fact has been scrutinized through the nanobody-containing fusion proteins EM1-GFP-H6 and A3C8-GFP-H6, whose biosynthesis results in two distinguishable populations of building blocks. In one of them, the assembling and disassembling is controllable by cations. However, a second population immediately self-assembles upon purification through a non-regulatable pathway, rendering larger nanoparticles with specific biological properties. The structural analyses of both model proteins and nanoparticles revealed important conformational variability in the building blocks. This fact renders different structural and functional categories of the final soft materials resulting from the participation of energetically unstable intermediates in the oligomerization process. These data illustrate the complexity of the Hismediated protein assembling in recombinant proteins but they also offer clues for a better design and refinement of protein-based nanomedicines, which, resulting from biological fabrication, show an architectonic flexibility unusual among biomaterials.

Formation of Natural Egg Yolk Granule Stabilized Pickering High Internal Phase Emulsions by Means of NaCl Ionic Strength and pH Change.

Pickering high internal phase emulsions (HIPEs) are gel-like concentrated emulsions that have the potential to be an alternative to partially hydrogenated oil (PHO). In this study, egg yolk granules (EYGs), natural complexes of protein and lipid isolated from egg yolk, were used as an emulsifier to prepare Pickering HIPEs. Gel-like HIPEs with an oil phase volume fraction of 85% and with an emulsifier concentration of only 0.5% could be prepared by using EYGs as an emulsifier. The EYGs were able to form stable HIPEs at NaCl ionic strengths over 0.2 M and at pH over 5.0 with NaCl ionic strength of 0.3 M. The EYGs, which could stabilize HIPEs, were easily to adsorb and cover the oil-water interface to form emulsion droplets with small particle size. In addition, interacting EYGs in the aqueous phase formed a continuous network structure, and the oil droplets packed closely, exhibiting high elasticity and shear thinning behavior. Furthermore, the formed HIPEs had suitable storage stability with no significant changes in appearance and microstructure after storage for 60 days. This work can transform traditional oils from liquid-like to solid-like by using EYGs to enrich food processing diversity and improve the storage stability of oils while reducing the intake of PHO and providing a healthier diet for consumers.

To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana.

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

Breast Cancer Treatment: The Case of Gold(I)-Based Compounds as a Promising Class of Bioactive Molecules.

Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6–7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA—therefore extensively studied—is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.

Polymolecular Complexes of Chitosan with the Bombyx Mori Protein

The interaction of chitosan (ChS) and the Bombyx mori protein on different pH ranges was studied, and the fundamental possibility of obtaining complexes of ChS with the Bombyx mori protein was revealed. The for-mation of a polymolecular complex of protein with ChS in aqueous solutions was confirmed by the results of physico-chemical methods. It is shown that the ChS structure is characterized by a certain rigidity and ionogenicity. The results indicate the complexation of the pupae protein with ChS in 2% acetic acid in the range of pH = 4.8–6.7. The detected changes and shifts of the absorption bands in the IR spectra confirm the occurrence of the complex formation reaction between the molecules of ChS and protein at pH = 4.8–6.7, which is characterized by absorption bands in the IR spectra at 1641 cm–1, 1538 cm–1 and 1068 cm–1. Quan-tum-chemical DFT study of ChS complexes with amino acids (AAs) was carried out. The stability of com-plexes of ChS with AAs (ChS-AA) was shown except for the complex formed with histidine in the gas phase. The calculation results indicate the presence of a strong thermodynamic driving force in the complexation of ChS with AAs.

Bioactive components of peppermint (Mentha piperita L.), their pharmacological and ameliorative potential and ethnomedicinal benefits: A review

Peppermint is a medicinal herb, cultivated globally and is known for its distinct flavour and aromatic fragrance. Peppermint is used in different industrial purposes, also consumed as tisane Peppermint is used as traditional medicine from time immemorial. Peppermint possesses flavonoids, volatile oils, lutolin, hesperidins etc. The major components of the volatile oil are methanol and methone. Peppermint has some efficacy as an antineoplastic, anti-microbial, anti-viral, anti-radiation, anti-oxidant as well as anti-inflammatory. Peppermint leaf is used mainly to treat Upper G.I tract diseases, respiratory tract problems, irritable bowel syndrome (IBS), nausea, hepatotoxicity, diabetes etc. Anti-microbial effect of peppermint mainly in corona virus (Covid-19) has been found also, peppermint helps to prevent the interaction between corona virus (Covid-19) and the host body by inhibiting the formation of receptor complex of viral spike protein and ACE2 receptors of the host body. Furthermore it may act directly on virus or may pierce the viral membrane or may inactivates essential viral enzymes. Its effectivity has also been seen during post-covid phase. The consumption of peppermint is generally safe for all age –groups, but intake of large volume can cause heart burn and gastroesophageal reflux disease (GERD). This article holistically reviews the therapeutic roles of peppermint in various physiological and disease conditions.

Alpha-Fetoprotein Binds Toxins and Can Be Used to Treat Cancer

The inefficiency of the immune system to kill cancer cells leads to the disease. Like in pregnancy, oncofetal proteins counteract the immune attack. Alpha-fetoprotein is an immunosuppressive protein generated by the embryo and in insignificant amounts by adults. It delivers nutrients to embryo cells and to the monocytes that suppress both innate and adaptive immunity during pregnancy and cancer. The small subpopulation of suppressor monocytes and cancer cells absorb the alpha-fetoprotein-nutrient complex through the specific receptor that is mostly absent in normal adult cells. It comes out that suppressor monocytes are the main targets in cancer prophylactic or treatment, not cancer cells. By delivering toxins instead of nutrients alpha-fetoprotein kills suppressor monocytes canceling immune suppression, as well as killing cancer cells directly. It is the perfect synergy of the most powerful cancer immunotherapy with targeted chemotherapy. Alpha-fetoprotein chemical conjugates, as well as the complexes of this oncofetal protein with binding toxins, have shown promising results in cancer treatments. Oral porcine alpha-fetoprotein complexes with toxins can prevent and/or treat cancer, although their immunotherapy mechanism of action is undiscovered.

INHIBITOR OF APOPTOSIS XIAP AND GLIOBLASTOMA CELLS RESISTANCE TO VACCINIA VIRUS VV-GMCSF-LACT

It has been shown that under the action VV-GMCSF-Lact, the death of glioblastoma cells occurs mainly through the apoptotic pathway. When analyzing the complexes of Smac protein with the apoptosis inhibitor XIAP in human glioblastoma cells, it was found that in the cells most sensitive to VV-GMCSF-Lact, Smac level was increased after incubation with the virus. At the same time, overall level of XIAP was higher in more resistant cells.