As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.
Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism* ; Golgi Apparatus/metabolism* ; Metabolic Engineering ; Vacuoles/metabolism*

Protein transport from endoplasmic reticulum (ER) to Golgi apparatus has long been known to be a central process for protein quality control and sorting. Recent studies have revealed that a large number of signal molecules are involved in regulation of membrane trafficking through ER, ER-Golgi intermediate compartment and Golgi apparatus. These molecules can significantly change the transport rate of proteins by regulating vesicle budding and fusion. Protein transport from ER to Golgi apparatus is not only controlled by signal pathways triggered from outside the cell, it is also regulated by feedback signals from the transport pathway.
Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Humans ; Protein Transport ; physiology ; Secretory Pathway ; Signal Transduction

Animals ; Cytoplasm ; metabolism ; Endoplasmic Reticulum, Rough ; metabolism ; Golgi Apparatus ; metabolism ; Humans ; Protein Transport ; Ricin ; chemistry ; pharmacokinetics ; therapeutic use

Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Golgi Apparatus ; genetics ; metabolism ; Golgi Matrix Proteins ; Insulin-Secreting Cells ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proinsulin ; genetics ; metabolism ; Rats ; rab1 GTP-Binding Proteins ; genetics ; metabolism

OBJECTIVETo compare the Golgi proteome of hepatocellular carcinoma (HCC) with that of the adjacent non-tumor tissues.

METHODSHepatocellular carcinoma and adjacent non-tumor tissues were obtained from HCC patients. The protein expression maps in Golgi were obtained by two-dimensional gel electrophoresis (2-DE), and the differentially expressed protein spots were analyzed by PD-Quest software. Peptide mass fingerprint (PMF) of differential protein spots was obtained with MALD-TOT-MS.

RESULTSAccording to 2-DE maps, the average numbers of protein spots were (1153+/-49) and (1086+/-37) in hepatocellular carcinoma and the adjacent non-tumor tissues. Compared to the adjacent non-tumor tissues, 27 proteins were upregulated, and 20 proteins were downregulated in HCC Golgi.

CONCLUSIONSThe Golgi proteome in HCC tissues is different from that in the adjacent non-tumor tissues, and the differential expression proteins are involved in energy metabolism, tumor metastasis, and cell cycle regulation.

Annexin A5 ; analysis ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; methods ; Golgi Apparatus ; metabolism ; Humans ; Liver ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Proteins ; analysis ; metabolism ; Proteome ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
Animals ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74-96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.
Amino Acid Sequence ; Animals ; Cells, Cultured ; Cytoplasmic Dyneins/chemistry/*metabolism ; Dendrites/metabolism ; Golgi Apparatus/metabolism ; HEK293 Cells ; Hippocampus ; Humans ; Molecular Sequence Data ; Neurons/*metabolism ; Phosphotransferases (Alcohol Group Acceptor)/*metabolism ; Protein Interaction Maps ; Rats, Sprague-Dawley ; Tubulin

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (C×31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of C×31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type C×31 protein (C×31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated C×31 mutants, C×31R180X and C×31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of C×31 mutants but not C×31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated C×31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated C×31 mutations.
Animals ; Connexins ; genetics ; Ear, Inner ; metabolism ; Endoplasmic Reticulum ; physiology ; Gap Junctions ; genetics ; metabolism ; physiology ; Golgi Apparatus ; genetics ; metabolism ; Hearing Loss ; genetics ; metabolism ; pathology ; Mice ; Mutation ; Neurons ; metabolism ; Protein Transport ; genetics ; Stress, Physiological

Objective: To investigate the clinical value of plasma scaffold protein SEC16A level and related models in the diagnosis of hepatitis B virus-related liver cirrhosis (HBV-LC) and hepatocellular carcinoma (HBV-HCC). Methods: Patients with HBV-LC and HBV-HCC and a healthy control group diagnosed by clinical, laboratory examination, imaging, and liver histopathology at the Third Hospital of Hebei Medical University between June 2017 and October 2021 were selected. Plasma SEC16A level was detected using an enzyme-linked immunosorbent assay (ELISA). Serum alpha-fetoprotein (AFP) was detected using an electrochemiluminescence instrument. SPSS 26.0 and MedCalc 15.0 statistical software were used to analyze the relationship between plasma SEC16A levels and the occurrence and development of liver cirrhosis and liver cancer. A sequential logistic regression model was used to analyze relevant factors. SEC16A was established through a joint diagnostic model. Receiver operating characteristic curve was used to evaluate the clinical efficacy of the model for liver cirrhosis and hepatocellular carcinoma diagnosis. Pearson correlation analysis was used to identify the influencing factors of novel diagnostic biomarkers. Results: A total of 60 cases of healthy controls, 60 cases of HBV-LC, and 52 cases of HBV-HCC were included. The average levels of plasma SEC16A were (7.41 ± 1.66) ng/ml, (10.26 ± 1.86) ng/ml, (12.79 ± 1.49) ng /ml, respectively, with P < 0.001. The sensitivity and specificity of SEC16A in the diagnosis of liver cirrhosis and hepatocellular carcinoma were 69.44% and 71.05%, and 89.36% and 88.89%, respectively. SEC16A, age, and AFP were independent risk factors for the occurrence of HBV-LC and HCC. SAA diagnostic cut-off values, sensitivity, and specificity were 26.21 and 31.46, 77.78% and 81.58%, and 87.23% and 97.22%, respectively. The sensitivity and specificity for HBV-HCC early diagnosis were 80.95% and 97.22%, respectively. Pearson correlation analysis showed that AFP level was positively correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and γ-glutamyltransferase (GGT) with P < 0.01, while the serum SEC16A level was only slightly positively correlated with ALT and AST in the liver cirrhosis group (r = 0.268 and 0.260, respectively, P < 0.05). Conclusion: Plasma SEC16A can be used as a diagnostic marker for hepatitis B-related liver cirrhosis and hepatocellular carcinoma. SEC16A, combined with age and the AFP diagnostic model with SAA, can significantly improve the rate of HBV-LC and HBV-HCC early diagnosis. Additionally, its application is helpful for the diagnosis and differential diagnosis of the progression of HBV-related diseases.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; alpha-Fetoproteins/metabolism* ; Endoplasmic Reticulum/metabolism* ; Golgi Apparatus/metabolism* ; Vesicular Transport Proteins ; Liver Cirrhosis/complications* ; Hepatitis B/complications* ; ROC Curve ; Hepatitis B virus/metabolism* ; Biomarkers, Tumor

A major advance in the understanding of the regulation of food intake has been the discovery of the adipokine leptin a hormone secreted by the adipose tissue. After crossing the blood-brain barrier, leptin reaches its main site of action at the level of the hypothalamic cells where it plays fundamental roles in the control of appetite and in the regulation of energy expenditure. At first considered as a hormone specific to the white adipose tissue, it was rapidly found to be expressed by other tissues. Among these, the gastric mucosa has been demonstrated to secrete large amounts of leptin. Secretion of leptin by the gastric chief cells was found to be an exocrine secretion. Leptin is secreted towards the gastric lumen into the gastric juice. We found that while secretion of leptin by the white adipose tissue is constitutive, secretion by the gastric cells is a regulated one responding very rapidly to secretory stimuli such as food intake. Exocrine-secreted leptin survives the hydrolytic conditions of the gastric juice by forming a complex with its soluble receptor. This soluble receptor is synthesized by the gastric cells and the leptin-leptin receptor complex gets formed at the level of the gastric chief cell secretory granules before being released into the gastric lumen. The leptin-leptin receptor upon resisting the hydrolytic conditions of the gastric juice is channelled, to the duodenum. Transmembrane leptin receptors expressed at the luminal membrane of the duodenal enterocytes interact with the luminal leptin. Leptin is actively transcytosed by the duodenal enterocytes. From the apical membrane it is transferred to the Golgi apparatus where it binds again its soluble receptor. The newly formed leptin-leptin receptor complex is then secreted baso-laterally into the intestinal mucosa to reach the blood capillaries and circulation thus reaching the hypothalamus where its action regulates food intake. Exocrine-secreted gastric leptin participates in the short term regulation of food intake independently from that secreted by the adipose tissue. Adipose tissue leptin on the other hand, regulates in the long term energy storage. Both tissues work in tandem to ensure management of food intake and energy expenditure.
Adipokines ; Adipose Tissue ; Adipose Tissue, White ; Appetite ; Blood-Brain Barrier ; Capillaries ; Chief Cells, Gastric ; Dietary Sucrose ; Duodenum ; Eating ; Energy Metabolism ; Enterocytes ; Gastric Juice ; Gastric Mucosa ; Golgi Apparatus ; Hand ; Hypothalamus ; Intestinal Mucosa ; Leptin ; Membranes ; Phenobarbital ; Receptors, Leptin ; Secretory Vesicles