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Applied Microbiology and Biotechnology Jun 2021Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella... (Review)
Review
Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell's physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia's potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. KEY POINTS: • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.
Topics: Fungal Proteins; Pichia; Recombinant Proteins; Saccharomycetales; Unfolded Protein Response
PubMed: 34037840
DOI: 10.1007/s00253-021-11336-5 -
Toxins Feb 2021Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell... (Review)
Review
Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).
Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cell-Penetrating Peptides; Drug Carriers; Humans; Venoms
PubMed: 33671927
DOI: 10.3390/toxins13020147 -
Pharmaceutical Biology Dec 2023Therapeutic effects of Qiangjing tablets (QJT) on sperm vitality and asthenozoospermia (AZS) have been confirmed. However, the mechanism of action remains unclear.
CONTEXT
Therapeutic effects of Qiangjing tablets (QJT) on sperm vitality and asthenozoospermia (AZS) have been confirmed. However, the mechanism of action remains unclear.
OBJECTIVE
This study investigates the effects of QJT on AZS and the underlying mechanism of action.
MATERIALS AND METHODS
Sixty Sprague-Dawley rats were randomly divided into six groups: Control, ORN (ornidazole; 200 mg/kg), ORN + QJT-low (0.17 g/mL), ORN + QJT-middle (0.33 g/mL), ORN + QJT-high (0.67 g/mL), and ORN + QJT + Radicicol (0.67 g/mL QJT and 20 mg/kg radicicol) groups. Pathological evaluation and analysis of mitophagy were conducted by H&E staining and transmission electron microscopy, respectively. Reactive oxygen species were detected by flow cytometry. Protein expression was determined by Western blotting.
RESULTS
QJT significantly improved ORN-treated sperm motility and kinematic parameters, as well as the pathological symptoms of testicular and epididymal tissues. In particular, QJT mitigated impaired mitochondrial morphology, and increased the PHB, Beclin-1, LC3-II protein, and ROS levels ( < 0.05), and reduced the protein expression levels of LC3-I and p62 ( < 0.05). Mechanistically, QJT antagonized the downregulation of SCF and Parkin protein levels ( < 0.05). Furthermore, QJT significantly increased the protein expressions levels of LKB1, AMPKα, p-AMPKα, ULK1 and p-ULK1 ( < 0.05). The ameliorative effect of QJT on pathological manifestations, mitochondrial morphology, and the expressions of mitophagy and mitochondrial ubiquitination-related proteins was counteracted by radicicol.
DISCUSSION AND CONCLUSIONS
QJT improved AZS mitochondrial ubiquitination and mitophagy mediated by the LKB1/AMPK/ULK1 signaling pathway. Our study provides a theoretical basis for the treatment of AZS and male infertility.
Topics: Animals; Male; Rats; AMP-Activated Protein Kinases; Asthenozoospermia; Autophagy-Related Protein-1 Homolog; Drugs, Chinese Herbal; Intracellular Signaling Peptides and Proteins; Mitophagy; Rats, Sprague-Dawley; Semen; Sperm Motility; Tablets; Ubiquitination
PubMed: 36655371
DOI: 10.1080/13880209.2023.2168021 -
Journal of Dental Research Jun 2020The oral microbiome is one of the most stable ecosystems in the body and yet the reasons for this are still unclear. As well as being stable, it is also highly diverse... (Review)
Review
The oral microbiome is one of the most stable ecosystems in the body and yet the reasons for this are still unclear. As well as being stable, it is also highly diverse which can be ascribed to the variety of niches available in the mouth. Previous studies have focused on the microflora in disease-either caries or periodontitis-and only recently have they considered factors that maintain the normal microflora. This has led to the perception that the microflora proliferate in nutrient-rich periods during oral processing of foods and drinks and starves in between times. In this review, evidence is presented which shows that the normal flora are maintained on a diet of salivary factors including urea, lactate, and salivary protein degradation. These factors are actively secreted by salivary glands which suggests these factors are important in maintaining normal commensals in the mouth. In addition, the immobilization of SIgA in the mucosal pellicle indicates a mechanism to retain certain bacteria that does not rely on the bacterial-centric mechanisms such as adhesins. By examining the salivary metabolome, it is clear that protein degradation is a key nutrient and the availability of free amino acids increases resistance to environmental stresses.
Topics: Dental Pellicle; Immunoglobulin A, Secretory; Microbiota; Mouth; Saliva; Salivary Proteins and Peptides
PubMed: 32283990
DOI: 10.1177/0022034520915486 -
Molecular and Cellular Endocrinology Jan 2020Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational... (Review)
Review
Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Those that fail must be recognized and eliminated to maintain ER homeostasis. Two cellular mechanisms have been identified to rid the ER of terminally unfolded, misfolded, and aggregated proteins. ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. ER-phagy has been more recently described and caters to larger, more complex proteins and protein aggregates that are not readily handled by ERAD. This pathway has unique upstream components and relies on the same downstream effectors of autophagy used in other cellular processes to deliver clients to lysosomes for degradation. In this review, we describe the main elements of ERQC, ERAD, and ER-phagy and focus on recent advances in these fields.
Topics: Animals; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Humans; Protein Folding; Protein Transport; Proteins; Secretory Pathway
PubMed: 31669350
DOI: 10.1016/j.mce.2019.110630 -
Advanced Drug Delivery Reviews May 2022The secreted mucus layer that lines and protects epithelial cells is conserved across diverse species. While the exact composition of this protective layer varies... (Review)
Review
The secreted mucus layer that lines and protects epithelial cells is conserved across diverse species. While the exact composition of this protective layer varies between organisms, certain elements are conserved, including proteins that are heavily decorated with N-acetylgalactosamine-based sugars linked to serines or threonines (O-linked glycosylation). These heavily O-glycosylated proteins, known as mucins, exist in many forms and are able to form hydrated gel-like structures that coat epithelial surfaces. In vivo studies in diverse organisms have highlighted the importance of both the mucin proteins as well as their constituent O-glycans in the protection and health of internal epithelia. Here, we summarize in vivo approaches that have shed light on the synthesis and function of these essential components of mucus.
Topics: Epithelial Cells; Glycosylation; Humans; Mucins; Mucus; Polysaccharides
PubMed: 35278522
DOI: 10.1016/j.addr.2022.114182 -
Human Vaccines & Immunotherapeutics Apr 2022In addition to the classical immunological functions such as neutralization, antibody-dependent cellular cytotoxicity, and complement activation, IgG antibodies possess... (Review)
Review
In addition to the classical immunological functions such as neutralization, antibody-dependent cellular cytotoxicity, and complement activation, IgG antibodies possess a little-recognized and under-utilized effector function at mucosal surfaces: trapping pathogens in mucus. IgG can potently immobilize pathogens that otherwise readily diffuse or actively swim through mucus by forming multiple low-affinity bonds between the array of pathogen-bound antibodies and the mucin mesh. Trapping in mucus can exclude pathogens from contacting target cells, and facilitate their rapid elimination by natural mucus clearance mechanisms. Despite the fact that most infections are transmitted at mucosal surfaces, this muco-trapping effector function has only been revealed within the past decade, with the evidence to date suggesting that it is a universal effector function of IgG-Fc capable of immobilizing both viral and highly motile bacterial pathogens in all major mucosal secretions. This review provides an overview of the current evidence for Fc-mucin crosslinking as an effector function for antibodies in mucus, the mechanism by which the accumulation of weak Fc-mucin bonds by IgG bound to the surface of a pathogen can result in immobilization of antibody-pathogen complexes, and how trapping in mucus can contribute to protection against foreign pathogens.
Topics: Antibody-Dependent Cell Cytotoxicity; Immunoglobulin G; Mucins; Mucus
PubMed: 34314289
DOI: 10.1080/21645515.2021.1939605 -
BMC Biology Jul 2022Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The...
BACKGROUND
Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown.
RESULTS
We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133.
CONCLUSIONS
Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
Topics: Animals; Fertility; Male; Mice; Semen; Testis; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35831855
DOI: 10.1186/s12915-022-01368-2 -
Biochimica Et Biophysica Acta. General... Jul 2020In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a β-barrel that aids...
BACKGROUND
In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted β-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the β-barrel domain of the autotransporter.
METHODS
To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA.
RESULTS
We observed that each protein's β-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's β-barrel is more than that through the BamA β-barrel.
CONCLUSIONS
Secretion of autotransporters most likely occurs through an incompletely formed β-barrel domain of the autotransporter in conjunction with BamA.
GENERAL SIGNIFICANCE
Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.
Topics: Bacterial Outer Membrane Proteins; Biological Transport; Escherichia coli; Escherichia coli Proteins; Humans; Models, Molecular; Protein Folding; Serine Endopeptidases; Type V Secretion Systems
PubMed: 32114025
DOI: 10.1016/j.bbagen.2020.129581 -
Journal of Chromatography. B,... Dec 2020Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion... (Review)
Review
Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.
Topics: Animals; Arthropods; Chromatography, Liquid; Drug Discovery; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry; Proteome; Proteomics; Snails; Snakes; Venoms
PubMed: 32971366
DOI: 10.1016/j.jchromb.2020.122352