Secretion Signal Research Articles

The Nβ motif of NaTrxh directs secretion as an endoplasmic reticulum transit peptide and variations might result in different cellular targeting.

Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the signal recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nβ, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h, with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues in plant proteins-a negative residue in position 8 is suggested in animal proteins-to maintain the Nβ motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nβ motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.

A monoclonal antibody targeting spore wall protein 1 inhibits the proliferation of Nosema bombycis in Bombyx mori.

Microsporidia are obligate intracellular single-cell eukaryotic parasites that can infect almost all kinds of animals and cause microsporidiosis. However, there are a few achievements of microsporidiosis treatments. Nosema bombycis could cause a microsporidiosis condition, pébrine, in the silkworm. Treatment and prevention of pébrine are still the research hotspots in sericulture. In this study, N. bombycis was treated with K2CO3, the spore walls were removed by centrifugation, and then, the alkali-soluble germination proteins were used as antigens to develop monoclonal antibodies (mAbs). Three mAbs were successfully screened and one mAb named G9 showed the highest titer among these monoclonal antibodies after enzyme-linked-immunosorbent serologic assay (ELISA). Mass spectrometry analysis confirmed that the mAb G9 could recognize the N. bombycis spore wall protein 1. Furthermore, the heavy chain and light chain sequences of the G9 monoclonal antibody were cloned, respectively. The vectors that expressing the intact antibodies and the single-chain variable fragments (scFvs) of G9 were constructed, and then, these vectors were used to develop the transgenic silkworm cell lines or transgenic silkworms. The inhibitory effects against N. bombycis were evaluated by the count of microsporidia and qPCR. The scFvs showed better effect on blocking the proliferation of N. bombycis than the intact antibody, and the scFv without the secretory signal peptide was more effective than that with signal peptide. Our study has provided novel strategies for microsporidiosis control and the essential groundwork for the future development of N. bombycis-resistant transgenic silkworms.IMPORTANCEThere are a few reports on the resistance of microsporidia, including Nosema bombycis. Here, the alkali-soluble germination proteins of N. bombycis were used as immunogens to prepare a monoclonal antibody, and its single-chain variable fragments effectively blocked microsporidia infection. Our study has provided novel strategies for microsporidiosis control and demonstrated a useful method for the potential treatment of other microsporidia diseases.

An unbiased proteomic analysis of PAD4 in human monocytes: novel substrates, binding partners and subcellular localizations.

Peptidylarginine deiminase IV (PAD4) post-translationally converts arginine residues in proteins to citrullines and is implicated in playing a central role in the pathogenesis of several diseases. Although PAD4 was historically thought to be a nuclear enzyme, recent evidence has revealed a more complex localization of PAD4 with evidence of additional cytosolic and cell surface localization and activity. However, the mechanisms by which PAD4, which lacks conventional secretory signal sequences, traffics to extranuclear localizations are unknown. In this study, we show that PAD4 was enriched in the organelle fraction of monocytes with evidence of citrullination of organelle proteins. We also demonstrated that PAD4 can bind to several cytosolic, nuclear and organelle proteins that may serve as binding partners for PAD4 to traffic intracellularly. Additionally, cell surface expression of PAD4 increased with monocyte differentiation into monocyte-derived dendritic cells and co-localized with several endocytic/autophagic and conventional secretory pathway markers, implicating the use of these pathways by PAD4 to traffic within the cell. Our results suggest that PAD4 is expressed in multiple subcellular localizations and may play previously unappreciated roles in physiological and pathological conditions. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

A multi-omics study to investigate the progression of the Correa pathway in gastric mucosa in the context of cirrhosis

BackgroundPatients with liver cirrhosis (LC) are prone to gastric mucosa damage. We investigated the alterations of gastric mucosa in LC patients and their possible mechanisms through multi-omics.ResultsWe observed significant gastric mucosa microbial dysbiosis in LC subjects. Gastric mucosal microbiomes of LC patients contained a higher relative abundance of Streptococcus, Neisseria, Prevotella, Veillonella, and Porphyromonas, as well as a decreased abundance in Helicobacter and Achromobacter, than control subjects. The LC patients had higher levels of bile acids (BAs) and long-chain acylcarnitines (long-chain ACs) in serum. The gastric mucosal microbiomes were associated with serum levels of BAs and long-chain ACs. Transcriptome analyses of gastric mucosa revealed an upregulation of endothelial cell specific molecule 1, serpin family E member 1, mucin 2, caudal type homeobox 2, retinol binding protein 2, and defensin alpha 5 in LC group. Besides, the bile secretion signaling pathway was significantly upregulated in the LC group.ConclusionsThe alterations in the gastric mucosal microbiome and transcriptome of LC patients were identified. The impaired energy metabolism in gastric mucosal cells and bile acids might aggravate the inflammation of gastric mucosa and even exacerbate the Correa’s cascade process. The gastric mucosal cells might reduce bile acid toxicity by bile acid efflux and detoxification.Trial registration: ChiCTR2100051070.

Ependymal cells SCOre sweet cerebrospinal fluid.

The subcommissural organ (SCO) is a secretory tissue located on the roof of the brain's third ventricle. A new study published in PLOS Biology finds that the SCO responds to glucose by secreting signaling molecules into the cerebrospinal fluid (CSF), thereby decreasing the local ependyma-driven CSF movement.

Development of novel recombinant peroxidase secretion system from Pseudomonas putida for lignin valorisation

Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin–depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.

Electrolyte imbalance causes suppression of NK and T cell effector function in malignant ascites

BackgroundMalignant ascites commonly occurs in advanced or recurrent stages of epithelial ovarian cancer during peritoneal carcinomatosis and is correlated with poor prognosis. Due to its complex composition of cellular and acellular components malignant ascites creates a unique tumor microenvironment, which mediates immunosuppression and promotes progression of disease. However, the immunosuppressive mechanisms remain poorly understood.MethodsIn the present study, we explored the antitumor activity of healthy donor NK and T cells directed against ovarian cancer cells in presence of malignant ascites derived from patients with advanced or recurrent peritoneal carcinomatosis. A wide range of methods was used to study the effect of ascites on NK and T cells (FACS, ELISA, EliSpot, qPCR, Live-cell and confocal microscopy, Western blot and electrolyte flux assays). The ascites components were assessed using quantitative analysis (nephelometry, potentiometry and clinical chemistry) and separation methods (dialysis, ultracentrifugal filtration and lipid depletion).ResultsAscites rapidly inhibited NK cell degranulation, tumor lysis, cytokine secretion and calcium signaling. Similarly, target independent NK and T cell activation was impaired in ascites environment. We identified imbalanced electrolytes in ascites as crucial factors causing extensive immunosuppression of NK and T cells. Specifically, high sodium, low chloride and low potassium content significantly suppressed NK-mediated cytotoxicity. Electrolyte imbalance led to changes in transcription and protein expression of electrolyte channels and impaired NK and T cell activation. Selected inhibitors of sodium electrolyte channels restored intracellular calcium flux, conjugation, degranulation and transcript expression of signaling molecules. The levels of ascites-mediated immunosuppression and sodium/chloride/potassium imbalance correlated with poor patient outcome and selected molecular alterations were confirmed in immune cells from ovarian cancer patients.ConclusionOur data suggest a novel electrolyte-based mechanism of immunosuppression in malignant ascites of patients with peritoneal carcinomatosis. We show for the first time that the immunosuppression of NK cytotoxicity in coculture assays is correlated to patient poor survival. Therapeutic application of sodium channel inhibitors may provide new means for restoring immune cell activity in ascites or similar electrolyte imbalanced environments.

Expression of secreted antimicrobial peptide in a marine diatom—Phaeodactylum tricornutum

Antimicrobial peptides (AMPs) are small-molecule polypeptides that occur widely in nature, have a broad antibacterial spectrum, and do not induce drug resistance in bacteria. Therefore, they are potential substitutes for antibiotics. However, there have been few reports of the production of the AMPs secreted by diatoms. Phaeodactylum tricornutum is a model diatom species for which protocols for its transformation are well established, and it can be directly used as fish and shrimp bait. In this study, we used P. tricornutum as a cell factory to produce secreted AMPs extracellularly. We also constructed an expression vector containing the secretory promoter and signal peptide of the Scygonadin gene, and successfully expressed AMPs in P. tricornutum, confirmed at the levels of DNA, RNA, and protein. Bacteriostatic experiments showed that the secreted AMPs exerted an obvious antibacterial effect on Kocuria sp. and Trelief5α Chemical Competent Cells. This study demonstrates that P. tricornutum effectively expresses and secretes AMPs with obvious antibacterial effects.

Interleukin-12 induces IFN-γ secretion and STAT signaling implying its potential regulation of Th1 cell response in Nile tilapia.

As a pleiotropic cytokine consisting of IL-12p35 and IL-12p40, Interleukin-12 (IL-12) features in inflammation regulation and anti-bacterial immunity. While IL-12 homologs have been identified in non-mammalian species, the precise mechanisms by which IL-12 contributes to early adaptive immune responses in vertebrates remain incompletely understood. Herein, an evolutionary conserved Oreochromis niloticus IL-12 (defined as OnIL-12) was identified by synteny characterization, structural comparisons and phylogenetic pattern of IL-12p35b and IL-12p40a. IL-12p35b and IL-12p40a exhibited widespread expression in lymphoid-related tissues of tilapia, while their mRNA expression in head-kidney demonstrated a significant increase after Edwardsiella piscicida infection. Compared with other lymphocytes, recombinant OnIL-12 (rOnIL-12) displayed stronger affinity binding to T cells. Although stimulation of lymphocytes with the p35b or p40a subunit resulted in a significant induction of IFN-γ expression, rOnIL-12 showed stronger potential to promote IFN-γ expression than these subunits. rOnIL-12 not only elevated the mRNA expression level Th1 cell-associated transcription factor T-bet in lymphocytes, but also increased the proportion of CD4-1+IFN-γ+ lymphocytes. Moreover, the mRNA and phosphorylation levels of STAT1, STAT3, STAT4 and STAT5 were enhanced by rOnIL-12. These findings will offer previous evidence for further exploration into the regulatory mechanisms of Th1 cellular immunity in early vertebrates.

Optimization of the proportions of advantageous components in the hypolipidemic “bioequivalent substance system” of Jiang-Zhi-Ning and its mechanism of action

Context Jiang-Zhi-Ning (JZN), a traditional Chinese medicinal formula, is used to treat hyperlipidemia in clinics. Objective To screen the hypolipidemic “bioequivalent substance system (BSS)” of JZN and elucidate the potential hypolipidemic mechanism. Materials and methods In vitro, the TG content in HepG2 cells was determined after the intervention of the combination of advantageous components (CAC) by uniform design. In vivo, hyperlipidemia models were established by Triton WR-1339 (400 mg/kg; i.p.) in male ICR mice, and corresponding treatments were administered via oral administration once. The mice were divided into 12 groups (n = 5): control, hyperlipidemic model, simvastatin (positive control, 20 mg/kg), gradient doses of JZN granules (2, 4 and 8 g/kg) and the hypolipidemic effective extraction (HEE) of JZN (120, 240 and 480 mg/kg) and CAC groups (20, 40 and 160 mg/kg). Serum TC, TG, LDL-C and HDL-C were performed after 24 h. Transcriptomics and qRT–PCR technology were used to explore the mechanism of the “BSS” of JZN. Results In vitro, the ratio of CAC was determined. CAC could reduce the TG content in HepG2 cells (77.21%). Compared with the model group, the high dose of CAC could markedly decrease the levels of TC (61.86%), TG (105.54%) and LDL-C (39.38%) and increase the level of HDL-C (232.67%). CAC was proved to be the “BSS”. Transcriptomics and qRT–PCR analysis revealed CAC regulated non-alcoholic fatty liver disease, bile secretion, PPAR and adipocytokine signalling pathway. Discussion and conclusions These findings provided new feasible ideas and methods for the elucidation of the pharmacodynamic material basis.

A new family of proteins is required for tethering of Casparian strip membrane domain and nutrient homoeostasis in rice.

Cell-cell junctions are essential for multicellular organisms to maintain nutrient homoeostasis. A plant-type tight junction, the Casparian strip (CS)-Casparian strip membrane domain (CSD) that seals the paracellular space between adjacent endodermal cells, has been known for more than one hundred years. However, the molecular basis of this structure remains unknown. Here we report that a new family of proteins containing a glycine/alanine/proline-rich domain, a lectin domain and a secretory signal peptide (GAPLESS) mediates tethering of the plasma membrane to the CS in rice. The GAPLESS proteins are specifically localized in the CS of root endodermal cells, and loss of their functions results in a disabled cell-cell junction and disrupted nutrient homoeostasis. The GAPLESS protein forms a tight complex with OsCASP1 in the plasma membrane, thereby mediating the CS-CSD junction. This study provides valuable insights into the junctional complex of plant endodermal cells, shedding light on our understanding of nutrient homoeostasis in crops and the cell junctions of eukaryotes.

Circadian rhythms in colonic function.

A rhythmic expression of clock genes occurs within the cells of multiple organs and tissues throughout the body, termed "peripheral clocks." Peripheral clocks are subject to entrainment by a multitude of factors, many of which are directly or indirectly controlled by the light-entrainable clock located in the suprachiasmatic nucleus of the hypothalamus. Peripheral clocks occur in the gastrointestinal tract, notably the epithelia whose functions include regulation of absorption, permeability, and secretion of hormones; and in the myenteric plexus, which is the intrinsic neural network principally responsible for the coordination of muscular activity in the gut. This review focuses on the physiological circadian variation of major colonic functions and their entraining mechanisms, including colonic motility, absorption, hormone secretion, permeability, and pain signalling. Pathophysiological states such as irritable bowel syndrome and ulcerative colitis and their interactions with circadian rhythmicity are also described. Finally, the classic circadian hormone melatonin is discussed, which is expressed in the gut in greater quantities than the pineal gland, and whose exogenous use has been of therapeutic interest in treating colonic pathophysiological states, including those exacerbated by chronic circadian disruption.

The RIX domain defines a class of polymorphic T6SS effectors and secreted adaptors

Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition, host colonization, or when resisting predation. Identifying effectors is a challenging task, as they lack canonical secretion signals or universally conserved domains. Here, we identify a protein domain, RIX, that defines a class of polymorphic T6SS cargo effectors. RIX is widespread in the Vibrionaceae family and is located at N-termini of proteins containing diverse antibacterial and anti-eukaryotic toxic domains. We demonstrate that RIX-containing proteins are delivered via T6SS into neighboring cells and that RIX is necessary and sufficient for T6SS-mediated secretion. In addition, RIX-containing proteins can enable the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism. The identification of RIX-containing proteins significantly enlarges the repertoire of known T6SS effectors, especially those with anti-eukaryotic activities. Furthermore, our findings also suggest that T6SSs may play an underappreciated role in the interactions between vibrios and eukaryotes.

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

TMCO3, a Putative K+ :Proton Antiporter at the Golgi Apparatus, Is Important for Longitudinal Growth in Mice and Humans.

Isolated short stature, defined as short stature without any other abnormalities, is a common heterogeneous condition in children. Exome sequencing identified the homozygous nonsense variant c.1832G>A/p.(Trp611*) in TMCO3 in two sisters with isolated short stature. Radiological studies, biochemical measurements, assessment of the skeletal status, and three-dimensional bone microarchitecture revealed no relevant skeletal and bone abnormalities in both sisters. The homozygous TMCO3 variant segregated with short stature in the family. TMCO3 transcript levels were reduced by ~50% in leukocyte-derived RNA of both sisters compared with controls, likely due to nonsense-mediated mRNA decay. In primary urinary cells of heterozygous family members, we detected significantly reduced TMCO3 protein levels. TMCO3 is functionally uncharacterized. We ectopically expressed wild-type TMCO3 in HeLa and ATDC5 chondrogenic cells and detected TMCO3 predominantly at the Golgi apparatus, whereas the TMCO3W611* mutant did not reach the Golgi. Coordinated co-expression of TMCO3W611* -HA and EGFP in HeLa cells confirmed intrinsic instability and/or degradation of the mutant. Tmco3 is expressed in all relevant mouse skeletal cell types. Highest abundance of Tmco3 was found in chondrocytes of the prehypertrophic zone in mouse and minipig growth plates where it co-localizes with a Golgi marker. Knockdown of Tmco3 in differentiated ATDC5 cells caused reduced and increased expression of Pthlh and Ihh, respectively. Measurement of long bones in Tmco3tm1b(KOMP)Wtsi knockout mice revealed significant shortening of forelimbs and hindlimbs. TMCO3 is a potential member of the monovalent cation:proton antiporter 2 (CPA2) family. By in silico tools and homology modeling, TMCO3 is predicted to have an N-terminal secretory signal peptide, forms a dimer localized to the membrane, and is organized in a dimerization and a core domain. The core domain contains the CPA2 motif essential for K+ binding and selectivity. Collectively, our data demonstrate that loss of TMCO3 causes growth defects in both humans and mice. © 2023 American Society for Bone and Mineral Research (ASBMR).

Comparative analysis of the effect of nano‑copper and Cu2O treatment on Cryptocaryon irritans tomonts in large yellow croaker (Larimichthys crocea) farming

Cryptocarya irritans is an obligate parasite causing the “white spot disease.” This disease often leads to the death of a large number of cultured marine fish. This study aimed to compare the effects of cuprous oxide (Cu2O) and nano‑copper on the morphology and hatchability of tomonts to investigate the effects of copper derivatives on C. irritans tomonts. The potential mechanistic difference was also explored using the transcriptome. The results showed that Cu2O seriously damaged the internal cell structure of tomonts, and the hatching rate of the tomonts in the Cu2O-treated group was lower than that in the control group within 10 days. On the contrary, the effect of nano‑copper on tomonts was not significant. The cytoplasm of tomonts treated with nano‑copper dissolved, the internal structure was slightly loose, and the hatching rate of tomonts was even slightly higher than control group within 5 days. In the transcriptome analysis, 850 differentially expressed genes (DEGs), including 332 upregulated genes and 518 downregulated genes, were identified in the Cu2O-treated groups. However, only 56 DEGs, including 34 upregulated genes and 22 downregulated genes, were identified in the nano‑copper-treated groups. This indicated that the effect of Cu2O on tomonts was greater than nano‑copper. In the enrichment pathway analysis, DEGs in the Cu2O-treated group were mostly enriched in the ribosome and apoptosis pathways. Further analysis showed that genes (RPL18e, RPLP0, RPL10, RPL17, RPL26, RPL35, and RPL37) related to cell stress and defense were significantly upregulated and those (P53 and PKA) related to cell proliferation and development were downregulated, indicating that the killing effect was mainly mediated by different ribosomal and structural proteins. However, the DEGs (KCNMA1, CACNA1C, CNGA1) in the nano‑copper-treated group were enriched in the insulin secretion and cAMP signaling pathways, which were activate part of tomonts' innate immune response, completely different from those in the Cu2O-treated group. The results provided more information on the mechanism of killing parasites by copper derivatives.

Pyroptosis -related potential diagnostic biomarkers in steroid-induced osteonecrosis of the femoral head

PurposeSteroid-induced necrosis of the femoral head (SONFH) is a refractory orthopedic hip disease occurring in young and middle-aged people, with glucocorticoids being the most common cause. Previous experimental studies have shown that cell pyroptosis may be involved in the pathological process of SONFH, but its pathogenesis in SONFH is still unclear. This study aims to screen and validate potential pyroptosis-related genes in SONFH diagnosis by bioinformatics analysis to further elucidate the mechanism of pyroptosis in SONFH.MethodsThere were 33 pyroptosis-related genes obtained from the prior reviews. The mRNA expression was downloaded from GSE123568 dataset in the Gene Expression Omnibus (GEO) database, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples. The pyroptosis-related differentially expressed genes involved in SONFH were identified with “affy” and “limma” R package by intersecting the GSE123568 dataset with pyroptosis genes. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the pyroptosis-related differentially expressed genes involved in SONFH were conducted by “clusterProfiler” R package and visualized by “GOplot” R package. Then, the correlations between the expression levels of the pyroptosis-related differentially expressed genes involved in SONFH were confirmed with “corrplot” R package. Moreover, the protein–protein interaction (PPI) network was analysed by using GeneMANIA database. Next, The ROC curve of pyroptosis-related differentially expressed genes were analyzed by “pROC” R package.ResultsA total of 10 pyroptosis-related differentially expressed genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 20 upregulated genes and 10 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 10 pyroptosis-related differentially expressed genes involved in SONFH were particularly enriched in cysteine-type endopeptidase activity involved in apoptotic process, positive regulation of interleukin-1 beta secretion and NOD-like receptor signaling pathway. Correlation analysis revealed significant correlations among the 10 differentially expressed pyroptosis-related genes involved in SONFH. The PPI results demonstrated that the 10 pyroptosis-related differentially expressed genes interacted with each other. Compared to non-SONFH samples, these pyroptosis-related differentially expressed genes had good predictive diagnostic efficacy (AUC = 1.000, CI = 1.000–1.000) in the SONFH samples, and NLRP1 had the highest diagnostic value (AUC: 0.953) in the SONFH samples.ConclusionsThere were 10 potential pyroptosis-related differentially expressed genes involved in SONFH were identified via bioinformatics analysis, which might serve as potential diagnostic biomarkers because they regulated pyroptosis. These results expand the understanding of SONFH associated with pyroptosis and provide new insights to further explore the mechanism of action and diagnosis of pyroptosis associated in SONFH.

Effects of signal peptides on the secreted green fluorescent protein (GFP) expression by recombinant Saccharomyces cerevisiae

Recombinant proteins secreted by Saccharomyces cerevisiae have been widely applied in the food industry and biopharmaceuticals because of their valuable features. However, low-secreted protein production in S. cerevisiae is a pending challenge that has hampered applications based on S. cerevisiae. To improve the secreted protein expression in S. cerevisiae, it is important to develop an efficient cassette for the secretory expression of recombinant protein by S. cerevisiae. In this study, three cassettes were constructed in which a gene of interest was fused with genes coding for secretory signal peptides from AGA2, MFα and α-glucoamylase. Green fluorescent protein (GFP) was employed as a model protein to investigate the secretion of recombinant protein mediated by these signal peptides in S. cerevisiae. The secreted expression of GFP was detected by Western Blot and dot-blot analyses using a specific antibody against GFP which showed the strongest GFP levels from MFα signal-related cassette. Therefore, their cassettes could be employed for following studies on the secretion of some other proteins.

Antibody screening reveals antigenic proteins involved in Talaromyces marneffei and human interaction.

Talaromycosis is a fungal infection that generally affects immunocompromised hosts and is one of the most frequent systemic mycoses in HIV patients, especially in endemic areas such as Southeast Asia. Talaromyces marneffei, the causative agent of talaromycosis, grows as a mold in the environment but adapts to the human body and host niches by transitioning from conidia to yeast-like cells. Knowledge of the human host and T. marneffei interaction has a direct impact on the diagnosis, yet studies are still lacking. The morbidity and mortality rates are high in taloromycosis patients if the diagnosis and treatments are delayed. Immunogenic proteins are excellent candidates for developing detection tools. Previously, we identified antigenic proteins that were recognized by antibodies from talaromycosis sera. Three of these identified proteins have been previously characterized in detail, while the others have not been explored. To expedite the progress of antigen discovery, the complete list of antigenic proteins and their features was fully reported in this study. Functional annotation and Gene Ontology examination revealed that these proteins showed a high association with membrane trafficking. Further bioinformatics analyses were performed to search for antigenic protein characteristics, including functional domains, critical residues, subcellular localization, secretory signals, and epitope peptide sequences. Expression profiling of these antigenic encoding genes was investigated using quantitative real-time PCR. The results demonstrated that most genes were expressed at low levels in the mold form, but were highly upregulated in the pathogenic yeast phase, consistent with the antigenic role of these genes during the human-host interaction. Most transcripts accumulated in the conidia, suggesting a role during phase transition. The collection of all antigen-encoding DNA sequences described here is freely accessible at GenBank, which could be useful for the research community to develop into biomarkers, diagnostic tests, research detection tools, and even vaccines.

Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb’s ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76–458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.