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Protein Expression and Purification Oct 2023The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA...
The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.
Topics: Humans; Escherichia coli; Peptide Elongation Factor Tu; Mitochondrial Proteins; HEK293 Cells; Recombinant Proteins; Membrane Proteins
PubMed: 37329934
DOI: 10.1016/j.pep.2023.106322 -
Cell Communication and Signaling : CCS Sep 2023Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation and signal transduction, making it crucial to explore... (Review)
Review
Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation and signal transduction, making it crucial to explore these interaction networks to understand complex biological processes. Traditional methods such as affinity purification and yeast two-hybrid assays have been shown to have limitations, as they can only isolate high-affinity molecular interactions under nonphysiological conditions or in vitro. Moreover, these methods have shortcomings for organelle isolation and protein subcellular localization. To address these issues, proximity labeling techniques have been developed. This technology not only overcomes the limitations of traditional methods but also offers unique advantages in studying protein spatial characteristics and molecular interactions within living cells. Currently, this technique not only is indispensable in research on mammalian nucleoprotein interactions but also provides a reliable approach for studying nonmammalian cells, such as plants, parasites and viruses. Given these advantages, this article provides a detailed introduction to the principles of proximity labeling techniques and the development of labeling enzymes. The focus is on summarizing the recent applications of TurboID and miniTurbo in mammals, plants, and microorganisms. Video Abstract.
Topics: Animals; Proteins; Protein Interaction Maps; Mammals
PubMed: 37777761
DOI: 10.1186/s12964-023-01310-1 -
Protein Science : a Publication of the... Sep 2023Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or... (Review)
Review
Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.
Topics: Humans; Human Growth Hormone; Recombinant Proteins; Receptors, Somatotropin
PubMed: 37428391
DOI: 10.1002/pro.4727 -
Microbiology Spectrum Feb 2024Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these... (Review)
Review
Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.
Topics: Humans; Hepacivirus; Viral Nonstructural Proteins; Proteome; Cell Line; Hepatitis C; Apolipoproteins E; Apolipoproteins; DNA-Binding Proteins; DNA Repair Enzymes
PubMed: 38230952
DOI: 10.1128/spectrum.02562-22 -
Protein Science : a Publication of the... Jun 2024While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial...
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
Topics: Histidine; Polyphosphates; Nitrilotriacetic Acid; Recombinant Proteins; Humans; Proteins
PubMed: 38747394
DOI: 10.1002/pro.5021 -
PeerJ 2023The crown-of-thorns starfish (COTS; ) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea,...
BACKGROUND
The crown-of-thorns starfish (COTS; ) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea, numbness, vomiting, joint aches and sometimes paralysis. Small molecule saponins and the plancitoxin proteins have been implicated in COTS toxicity.
METHODS
Brine shrimp lethality assays were used to confirm the secretion of spine toxin biomolecules. Histological analysis, followed by spine-derived proteomics helped to explain the source and identity of proteins, while quantitative RNA-sequencing and phylogeny confirmed target gene expression and relative conservation, respectively.
RESULTS
We demonstrate the lethality of COTS spine secreted biomolecules on brine shrimp, including significant toxicity using aboral spine semi-purifications of >10 kDa ( > 0.05, 9.82 µg/ml), supporting the presence of secreted proteins as toxins. Ultrastructure observations of the COTS aboral spine showed the presence of pores that could facilitate the distribution of secreted proteins. Subsequent purification and mass spectrometry analysis of spine-derived proteins identified numerous secretory proteins, including plancitoxins, as well as those with relatively high gene expression in spines, including phospholipase A2, protease inhibitor 16-like protein, ependymin-related proteins and those uncharacterized. Some secretory proteins (, vitellogenin and deleted in malignant brain tumor protein 1) were not highly expressed in spine tissue, yet the spine may serve as a storage or release site. This study contributes to our understanding of the COTS through functional, ultrastructural and proteomic analysis of aboral spines.
Topics: Animals; Proteomics; Artemia; Arthralgia; Biological Assay; Biological Transport
PubMed: 37637177
DOI: 10.7717/peerj.15689 -
Journal of Translational Medicine Jul 2023Substantial studies have demonstrated that oxidative stress placenta and endothelial injury are considered to inextricably critical events in the pathogenesis of...
Small extracellular vesicles-transported lncRNA TDRKH-AS1 derived from AOPPs-treated trophoblasts initiates endothelial cells pyroptosis through PDIA4/DDIT4 axis in preeclampsia.
BACKGROUND
Substantial studies have demonstrated that oxidative stress placenta and endothelial injury are considered to inextricably critical events in the pathogenesis of preeclampsia (PE). Systemic inflammatory response and endothelial dysfunction are induced by the circulating factors released from oxidative stress placentae. As a novel biomarker of oxidative stress, advanced oxidation protein products (AOPPs) levels are strongly correlated with PE characteristics. Nevertheless, the molecular mechanism underlying the effect of factors is still largely unknown.
METHODS
With the exponential knowledge on the importance of placenta-derived extracellular vesicles (pEVs), we carried out lncRNA transcriptome profiling on small EVs (sEVs) secreted from AOPPs-treated trophoblast cells and identified upregulated lncRNA TDRKH-AS1 as a potentially causative factor for PE. We isolated and characterized sEVs from plasma and trophoblast cells by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. The expression and correlation of lncRNA TDRKH-AS1 were evaluated using qRT-PCR in plasmatic sEVs and placentae from patients. Pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs was performed to detect the TDRKH-AS1 function in vivo. To investigate the potential effect of sEVs-derived TDRKH-AS1 on endothelial function in vitro, transcriptome sequencing, scanning electron Microscopy (SEM), immunofluorescence, ELISA and western blotting were conducted in HUVECs. RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP) and coimmunoprecipitation (Co-IP) were used to reveal the latent mechanism of TDRKH-AS1 on endothelial injury.
RESULTS
The expression level of TDRKH-AS1 was significantly increased in plasmatic sEVs and placentae from patients, and elevated TDRKH-AS1 in plasmatic sEVs was positively correlated with clinical severity of the patients. Moreover, pregnant mice injected with TDRKH-AS1-riched trophoblast sEVs exhibited a hallmark feature of PE with increased blood pressure and systemic inflammatory responses. Pyroptosis, an inflammatory form of programmed cell death, is involved in the development of PE. Indeed, our in vitro study indicated that sEVs-derived TDRKH-AS1 secreted from AOPPs-induced trophoblast elevated DDIT4 expression levels to trigger inflammatory response of pyroptosis in endothelial cells through interacting with PDIA4.
CONCLUSIONS
Herein, results in the present study supported that TDRKH-AS1 in sEVs isolated from oxidative stress trophoblast may be implicated in the pathogenesis of PE via inducing pyroptosis and aggravating endothelial dysfunction.
Topics: Female; Pregnancy; Humans; Animals; Mice; Endothelial Cells; Pyroptosis; Advanced Oxidation Protein Products; RNA, Long Noncoding; Trophoblasts; Pre-Eclampsia; Extracellular Vesicles; RNA-Binding Proteins; Transcription Factors; Protein Disulfide-Isomerases
PubMed: 37488572
DOI: 10.1186/s12967-023-04346-6 -
Journal of Virology Sep 2023While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to...
While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation . IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.
Topics: Animals; Humans; Angiotensin-Converting Enzyme 2; Chiroptera; East Asian People; Longitudinal Studies; Receptors, Virus; Severe acute respiratory syndrome-related coronavirus; Spike Glycoprotein, Coronavirus; Trypsin; Betacoronavirus; Zoonoses
PubMed: 37655938
DOI: 10.1128/jvi.00395-23 -
Biomedicine & Pharmacotherapy =... Sep 2023All forms of life produce nanosized extracellular vesicles called exosomes, which are enclosed in lipid bilayer membranes. Exosomes engage in cell-to-cell communication... (Review)
Review
All forms of life produce nanosized extracellular vesicles called exosomes, which are enclosed in lipid bilayer membranes. Exosomes engage in cell-to-cell communication and participate in a variety of physiological and pathological processes. Exosomes function via their bioactive components, which are delivered to target cells in the form of proteins, nucleic acids, and lipids. Exosomes function as drug delivery vehicles due to their unique properties of innate stability, low immunogenicity, biocompatibility, biodistribution, accumulation in desired tissues, low toxicity in normal tissues, and the stimulation of anti-cancer immune responses, and penetration capacity into distance organs. Exosomes mediate cellular communications by delivering various bioactive molecules including oncogenes, oncomiRs, proteins, specific DNA, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), and circular RNA (circRNA). These bioactive substances can be transferred to change the transcriptome of target cells and influence tumor-related signaling pathways. After considering all of the available literature, in this review we discuss the biogenesis, composition, production, and purification of exosomes. We briefly review exosome isolation and purification techniques. We explore great-length exosomes as a mechanism for delivering a variety of substances, including proteins, nucleic acids, small chemicals, and chemotherapeutic drugs. We also talk about the benefits and drawbacks of exosomes. This review concludes with a discussion future perspective and challenges. We hope that this review will provide us a better understanding of the current state of nanomedicine and exosome applications in biomedicine.
Topics: Exosomes; Tissue Distribution; Drug Delivery Systems; MicroRNAs; Proteins; RNA, Small Interfering; Nucleic Acids; Neoplasms
PubMed: 37392659
DOI: 10.1016/j.biopha.2023.115087 -
Molecules (Basel, Switzerland) Aug 2023Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and... (Review)
Review
Among the toxic metabolites of the fungal world, those that, due to their strong biological effect, can seriously (even fatally) damage the life processes of humans (and certain groups of animals) stand out. Amatoxin-containing mushrooms and the poisonings caused by them stand out from the higher fungi, the mushrooms. There are already historical data and records about such poisonings, but scientific research on the responsible molecules began in the middle of the last century. The goals of this review work are as follows: presentation of the cosmopolitan mushroom species that produce amanitins (which are known from certain genera of four mushroom families), an overview of the chemical structure and specific properties of amanitins, a summary of the analytical methods applicable to them, a presentation of the "medical history" of poisonings, and a summary of the therapeutic methods used so far. The main responsible molecules (the amanitins) are bicyclic octapeptides, whose structure is characterized by an outer loop and an inner loop (bridge). It follows from the unusual properties of amanitins, especially their extreme stability (against heat, the acidic pH of the medium, and their resistance to human, and animal, digestive enzymes), that they are absorbed almost without hindrance and quickly transported to our vital organs. Adding to the problems is that accidental consumption causes no noticeable symptoms for a few hours (or even 24-36 h) after consumption, but the toxins already damage the metabolism of the target organs and the synthesis of nucleic acid and proteins. The biochemical catastrophe of the cells causes irreversible structural changes, which lead to necrotic damage (in the liver and kidneys) and death. The scientific topicality of the review is due to the recent publication of new data on the probable antidote molecule (ICR: indocyanine green) against amanitins. Further research can provide a new foundation for the therapeutic treatment of poisonings, and the toxicological situation, which currently still poses a deadly threat, could even be tamed into a controllable problem. We also draw attention to the review conclusions, as well as the mycological and social tasks related to amanitin poisonings (prevention of poisonings).
Topics: Amanitins; Agaricales; Humans; Animals; Mushroom Poisoning
PubMed: 37570902
DOI: 10.3390/molecules28155932