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Synthetic and Systems Biotechnology Dec 2024Collagen XVII (COL17) is a transmembrane protein that mediates skin homeostasis. Due to expression of full length collagen was hard to achieve in microorganisms, arising...
Collagen XVII (COL17) is a transmembrane protein that mediates skin homeostasis. Due to expression of full length collagen was hard to achieve in microorganisms, arising the needs for selection of collagen fragments with desired functions for microbial biosynthesis. Here, COL17 fragments (27-33 amino acids) were extracted and replicated 16 times for recombinant expression in . Five variants were soluble expressed, with the highest yield of 223 mg/L. The fusion tag was removed for biochemical and biophysical characterization. Circular dichroism results suggested one variant (sample-1707) with a triple-helix structure at >37 °C. Sample-1707 can assemble into nanofiber (width, 5.6 nm) and form hydrogel at 3 mg/mL. Sample-1707 was shown to induce blood clotting and promote osteoblast differentiation. Furthermore, sample-1707 exhibited high capacity to induce mouse hair follicle stem cells differentiation and osteoblast migration, demonstrating a high capacity to induce skin cell regeneration and promote wound healing. A strong hydrogel was prepared from a chitosan and sample-1707 complex with a swelling rate of >30 % higher than simply using chitosan. Fed-batch fermentation of sample-1707 with a 5-L bioreactor obtained a yield of 600 mg/L. These results support the large-scale production of sample-1707 as a biomaterial for use in the skin care industry.
PubMed: 38911060
DOI: 10.1016/j.synbio.2024.06.001 -
Plant Direct Jun 2024The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use...
The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of that we captured by a cyclic guanosine monophosphate (cGMP) affinity chromatography approach, namely, soluble starch synthase STA3, starch-branching enzyme SBE1, and α-amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzyme's N-terminal aspartate kinase-chorismate mutase-tyrA domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N and C metabolism at the enzymatic level, increasing the liberation of C skeletons from starch when high Gln levels signal an abundance of assimilated N.
PubMed: 38911017
DOI: 10.1002/pld3.609 -
Nature Communications Jun 2024Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to...
Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.
Topics: Escherichia coli; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Vibrio; Protein Biosynthesis; Gene Expression Regulation, Bacterial; RNA, Messenger; Clustered Regularly Interspaced Short Palindromic Repeats; Operon; Genetic Engineering; Lactic Acid
PubMed: 38909033
DOI: 10.1038/s41467-024-49642-x -
Nature Communications Jun 2024Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in...
Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in nasopharyngeal carcinoma remains to be elucidated. Here, we identify PJA1 as a key E3 ubiquitin ligase involved in nasopharyngeal carcinoma chemoresistance that is highly expressed in nasopharyngeal carcinoma patients with nonresponse to docetaxel-cisplatin-5-fluorouracil induction chemotherapy. We find that PJA1 facilitates docetaxel resistance by inhibiting GSDME-mediated pyroptosis in nasopharyngeal carcinoma cells. Mechanistically, PJA1 promotes the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, which further facilitates DRP1 phosphorylation at S637 and reduced mitochondrial reactive oxygen species production, resulting in suppression of GSDME-mediated pyroptosis and the antitumour immune response. PGAM5 knockdown fully restores the docetaxel sensitization effect of PJA1 knockdown. Moreover, pharmacological targeting of PJA1 with the small molecule inhibitor RTA402 enhances the docetaxel sensitivity of nasopharyngeal carcinoma in vitro and in vivo. Clinically, high PJA1 expression indicates inferior survival and poor clinical efficacy of TPF IC in nasopharyngeal carcinoma patients. Our study emphasizes the essential role of E3 ligases in regulating chemoresistance and provides therapeutic strategies for nasopharyngeal carcinoma based on targeting the ubiquitin-proteasome system.
Topics: Humans; Docetaxel; Drug Resistance, Neoplasm; Nasopharyngeal Carcinoma; Cell Line, Tumor; Nasopharyngeal Neoplasms; Pyroptosis; Ubiquitination; Animals; Ubiquitin-Protein Ligases; Mice; Mice, Nude; Female; Dynamins; Reactive Oxygen Species; Phosphoprotein Phosphatases; Male; Xenograft Model Antitumor Assays; Mice, Inbred BALB C; Antineoplastic Agents; Phosphorylation; Mitochondrial Proteins; Fluorouracil; Gene Expression Regulation, Neoplastic; Mitochondria; Cisplatin; Middle Aged; Gasdermins
PubMed: 38906860
DOI: 10.1038/s41467-024-49675-2 -
Science Advances Jun 2024The central nervous system coordinates peripheral cellular stress responses, including the unfolded protein response of the mitochondria (UPR); however, the contexts for...
The central nervous system coordinates peripheral cellular stress responses, including the unfolded protein response of the mitochondria (UPR); however, the contexts for which this regulatory capability evolved are unknown. UPR is up-regulated upon pathogenic infection and in metabolic flux, and the olfactory nervous system has been shown to regulate pathogen resistance and peripheral metabolic activity. Therefore, we asked whether the olfactory nervous system in controls the UPR cell nonautonomously. We found that silencing a single inhibitory olfactory neuron pair, AWC, led to robust induction of UPR and reduction of oxidative phosphorylation dependent on serotonin signaling and -mediated mitophagy. Further, AWC ablation confers resistance to the pathogenic bacteria partially dependent on the UPR transcription factor and fully dependent on mitophagy machinery. These data illustrate a role for the olfactory nervous system in regulating whole-organism mitochondrial dynamics, perhaps in preparation for postprandial metabolic stress or pathogenic infection.
Topics: Animals; Caenorhabditis elegans; Mitophagy; Mitochondria; Caenorhabditis elegans Proteins; Smell; Unfolded Protein Response; Pseudomonas aeruginosa; Ubiquitin-Protein Ligases; Oxidative Phosphorylation; Signal Transduction; Serotonin; Transcription Factors
PubMed: 38905346
DOI: 10.1126/sciadv.adn0014 -
International Journal of Biological... 2024Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of...
Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, and . Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress . Endothelium-specific knockout of DKK1 (DKK1/APOE) and endothelium-specific overexpression of DKK1 (DKK1) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation . RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1 and DKK1 mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1/APOE mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.
Topics: Animals; Atherosclerosis; Mice; Intercellular Signaling Peptides and Proteins; Foam Cells; Myocytes, Smooth Muscle; Endothelial Cells; Humans; Ubiquitination; Male; Coculture Techniques; Mice, Knockout; Ubiquitin-Specific Proteases; Mice, Inbred C57BL
PubMed: 38904030
DOI: 10.7150/ijbs.91957 -
Cureus May 2024Multiple congenital anomalies-hypotonia-seizures syndrome 3 (MCAHS3) results from mutations in the phosphatidylinositol glycan biosynthesis class T (PIGT) gene leading... (Review)
Review
Spectrum of Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (MCAHS3) Due to Phosphatidylinositol Glycan Biosynthesis Class T (PIGT) Gene Mutations: A Narrative Review.
Multiple congenital anomalies-hypotonia-seizures syndrome 3 (MCAHS3) results from mutations in the phosphatidylinositol glycan biosynthesis class T (PIGT) gene leading to defects in glycosylphosphatidylinositol transamidase complex (GPI-TA) synthesis. Glycosylphosphatidylinositol serves as an anchor to more than 150 mammalian proteins for attachment on cell surfaces, enabling specific functional properties. Mutations in the PIGT gene result in disruption of this extremely important post-translational protein modification, yielding dysfunctional proteins leading to MCAHS3. An exhaustive literature search was conducted across various electronic databases to reveal only 41 reported cases of MCAHS3 worldwide, emphasizing the rarity of this condition. Multiple congenital anomalies-hypotonia-seizures syndrome 3 has been reported as secondary to 18 different known PIGT variants to date, manifesting as a varying spectrum of craniofacial dysmorphism, developmental delay with epilepsy, cardiac and renal malformations, and unique features in biochemical testing and neuroimaging. This review aims to highlight the constellation of clinical symptoms, diagnostic modalities, and management challenges associated with MCAHS3 cases. It would help determine optimal diagnostic and treatment strategies for newly identified cases and facilitate new research on this rare condition.
PubMed: 38903302
DOI: 10.7759/cureus.60737 -
Disease Models & Mechanisms Jun 2024Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in...
Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9 simulating a CMS patient. As Gfpt1 exon 9 is exclusively included in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (i) UDP-HexNAc, CMP-NeuAc, and protein O-GlcNAcylations were reduced in skeletal muscles; (ii) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (iii) markers for unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of ER stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation, and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.
PubMed: 38903011
DOI: 10.1242/dmm.050768 -
Journal of Nanobiotechnology Jun 2024Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the...
BACKGROUND
Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.
RESULTS
In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.
CONCLUSIONS
Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome.
Topics: Humans; Spectrum Analysis, Raman; Receptor, ErbB-2; Breast Neoplasms; Cell Line, Tumor; Female; Gene Silencing; Metal Nanoparticles
PubMed: 38902746
DOI: 10.1186/s12951-024-02600-7 -
BMC Plant Biology Jun 2024The plant-specific YABBY transcription factor family plays important roles in plant growth and development, particularly leaf growth, floral organ formation, and...
BACKGROUND
The plant-specific YABBY transcription factor family plays important roles in plant growth and development, particularly leaf growth, floral organ formation, and secondary metabolite synthesis.
RESULTS
Here, we identified a total of 13 OfYABBY genes from the Osmanthus fragrans genome. These 13 OfYABBY genes were divided into five subfamilies through phylogenetic analysis, and genes in the same subfamily showed similar gene structures and conserved protein motifs. Gene duplication promoted the expansion of the OfYABBY family in O. fragrans. Tissue-specific expression analysis showed that the OfYABBY family was mainly expressed in O. fragrans leaves and floral organs. To better understand the role of OfYABBY genes in plant growth and development, OfYABBY12 was selected for heterologous stable overexpression in tobacco, and OfYABBY12-overexpressing tobacco leaves released significantly fewer volatile organic compounds than wild-type tobacco leaves. Overexpression of OfYABBY12 led to the downregulation of NtCCD1/4 and decreased β-ionone biosynthesis. Correspondingly, a dual-luciferase assay showed that OfYABBY12 negatively regulated the expression of OfCCD4, which promotes β-ionone synthesis. Furthermore, tobacco leaves overexpressing OfYABBY12 were curled and wrinkled and had significantly reduced leaf thickness and leaf inclusions and significantly extended flower pistils (styles).
CONCLUSION
Overall, the results suggest that the OfYABBY gene family may influence the biosynthesis of the floral scent (especially β-ionone) in O. fragrans and may regulate leaf morphogenesis and lateral organs.
Topics: Plant Leaves; Oleaceae; Flowers; Transcription Factors; Plant Proteins; Gene Expression Regulation, Plant; Phylogeny; Nicotiana; Odorants; Volatile Organic Compounds
PubMed: 38902627
DOI: 10.1186/s12870-024-05047-y