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Journal of Lipid Research Feb 2024Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density...
Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3 cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3 cells; rather ANGPTL3 cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3 cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3;LDLR cells rescued the deficient LDL clearance of LDLR cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.
Topics: Angiopoietin-Like Protein 3; Angiopoietin-like Proteins; Apolipoprotein B-100; Lipid Metabolism; Lipoproteins; Liver; Triglycerides
PubMed: 38219820
DOI: 10.1016/j.jlr.2024.100500 -
ELife Sep 2023The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium . Its environmental persistence provoking recurring...
The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium . Its environmental persistence provoking recurring sudden outbreaks is enabled by rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting survival and virulence. Our findings hold far-reaching implications for all strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.
Topics: Transcription Factors; DNA-Binding Proteins; Bacterial Proteins; Bile; Vibrio; Vibrio cholerae; Bile Acids and Salts; Gene Expression Regulation, Bacterial
PubMed: 37768326
DOI: 10.7554/eLife.88721 -
BMC Biology Oct 2023Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive...
BACKGROUND
Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis.
RESULTS
We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system.
CONCLUSIONS
Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.
Topics: Amylases; Bacterial Proteins; Bacillus subtilis; Adenosine Triphosphatases; Protein Transport; Cell Wall; Escherichia coli Proteins
PubMed: 37794427
DOI: 10.1186/s12915-023-01684-1 -
Journal of Biomolecular Structure &... Nov 2023Human breast milk contains free oligosaccharides (Human Milk Oligosaccharides-HMOs) that help to protect breastfed infants against a variety of infectious diseases and...
Human breast milk contains free oligosaccharides (Human Milk Oligosaccharides-HMOs) that help to protect breastfed infants against a variety of infectious diseases and act as decoy receptors. In breast milk, HMOs are the third most abundant compounds after lactose and lipids. Structural and conformational models of HMOs are quite crucial to studying the interaction with proteins and molecular recognition phenomenon. Molecular dynamics simulations for two trisaccharides HMOs (2'-FL and 3-FL) were carried out for 250 ns and the conformational models were subsequently substantiated by three replicate simulations. The conformer models of HMOs 2'-FL and 3-FL were deposited in the 3-Dimensional Structural Database for Sialic acid-containing CARbohydrates (3DSDSCAR) database website (www.3dsdscar.in). HMOs were then docked into the active site of norovirus capsid protein and are simulated for 100 ns duration. Each complex system was stabilized by direct and water-mediated hydrogen bonding interactions. Binding free energy calculations predict two possible binding modes for each complex system. The conformational flexibility and binding stability of the complex systems were calculated. The protein folding/unfolding and compactness seem to be better for the two HMOs. From a general perspective, we found that both 2'-FL and 3-FL exhibited higher binding efficacy towards norovirus capsid protein and according to the structural stability, 3-FL might be used as a preventive inhibitor for norovirus infection.Communicated by Ramaswamy H. Sarma.
Topics: Female; Humans; Capsid Proteins; Norovirus; Molecular Dynamics Simulation; Oligosaccharides; Milk, Human
PubMed: 36476051
DOI: 10.1080/07391102.2022.2153923 -
Frontiers in Immunology 2023The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of... (Review)
Review
The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of immune cell receptors. Although knowledge in the field of glycoimmunology is still limited, numerous studies point to the key role of glycosylation in maintaining homeostasis, but also in reflecting its disruption. Changes in oligosaccharide patterns can lead to impairment of both innate and acquired immune responses, with important implications in the pathogenesis of diseases, including autoimmunity. B cells appear to be unique within the immune system, since they exhibit both innate and adaptive immune activity. B cell surface is rich in glycosylated proteins and lectins which recognise glycosylated ligands on other cells. Glycans are important in the development, selection, and maturation of B cells. Changes in sialylation and fucosylation of cell surface proteins affect B cell signal transduction through BCRs, CD22 inhibitory coreceptor and Siglec-G. Plasmocytes, as the final stage of B cell differentiation, produce and secrete immunoglobulins (Igs), of which IgGs are the most abundant -glycosylated proteins in human serum with the conserved -glycosylation site at Asn297. -oligosaccharide composition of the IgG Fc region affects its secretion, structure, half-life and effector functions (ADCC, CDC). IgG -glycosylation undergoes little change during homeostasis, and may gradually be modified with age and during ongoing inflammatory processes. Hyperactivated B lymphocytes secrete autoreactive antibodies responsible for the development of autoimmunity. The altered profile of IgG -glycans contributes to disease progression and remission and is sensitive to the application of therapeutic substances and immunosuppressive agents. In this review, we focus on the role of -glycans in B-cell biology and IgG activity, the rearrangement of IgG oligosaccharides in aging, autoimmunity and immunosuppressive therapy.
Topics: Humans; Glycosylation; Immunoglobulin G; Autoimmunity; B-Lymphocytes; Polysaccharides; Oligosaccharides; Anti-Inflammatory Agents
PubMed: 37575234
DOI: 10.3389/fimmu.2023.1188838 -
Journal of Dairy Science Aug 2023Gastric digestion of 2 commercial ultrafiltered milks and milk enriched with skim milk powder (to simulate concentration by reverse osmosis) was investigated and...
Gastric digestion of 2 commercial ultrafiltered milks and milk enriched with skim milk powder (to simulate concentration by reverse osmosis) was investigated and compared with the digestion of nonconcentrated milk. Curd formation and proteolysis of high-protein milks in simulated gastric conditions were studied using oscillatory rheology, extrusion testing, and gel electrophoresis. The presence of pepsin in the gastric fluid triggered coagulation at pH >6 and the elastic modulus of gels from high-protein milks was ~5 times larger than the gel from reference milk. Despite similar protein concentrations, the coagulum from milk enriched with skim milk powder showed higher resistance to shear deformation than the coagula from ultrafiltered milks. The gel structure was also more heterogeneous. During digestion, the degradation of coagula from high-protein milks was slowed down compared with the coagulum from reference milk, and intact milk proteins were still detected after 120 min. Differences in the digestion patterns of coagula from high-protein milks were observed and were associated with the proportion of minerals bound to caseins and the denaturation rate of whey proteins.
Topics: Animals; Proteolysis; Powders; Milk; Milk Proteins; Whey Proteins; Caseins; Rheology; Hydrogen-Ion Concentration
PubMed: 37291035
DOI: 10.3168/jds.2022-22714 -
Journal of Proteome Research Sep 2023Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming...
Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor β1 (TGF-β1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-β1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-β1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-β1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-β1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-β1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-β1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-β1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-β1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-β1-driven fibrosis events in HSGs.
Topics: Humans; Aquaporin 5; Collagen Type VII; Saliva; Submandibular Gland; Transforming Growth Factor beta1
PubMed: 37549151
DOI: 10.1021/acs.jproteome.3c00052 -
Proceedings of the National Academy of... Aug 2023Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited....
Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.
Topics: Plasmodium falciparum; Protozoan Proteins; Plasmodium; Protein Transport; Biological Transport; Erythrocytes
PubMed: 37552754
DOI: 10.1073/pnas.2308676120 -
Diabetes, Obesity & Metabolism Sep 2023Glucagon-like peptide-1 receptor agonists provide multiple benefits to patients with type 2 diabetes, including improved glycaemic control, weight loss and decreased...
AIM
Glucagon-like peptide-1 receptor agonists provide multiple benefits to patients with type 2 diabetes, including improved glycaemic control, weight loss and decreased risk of major adverse cardiovascular events. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses.
METHODS
Exenatide (5 μg, subcutaneously) or saline (0.2 ml, subcutaneously) was administered to 62 healthy volunteers. Frequently sampled intravenous glucose tolerance tests were conducted to assess the impact of exenatide on insulin secretion and insulin action. This pilot study was a crossover design in which participants received exenatide and saline in random order.
RESULTS
Exenatide increased first phase insulin secretion 1.9-fold (p = 1.9 × 10 ) and accelerated the rate of glucose disappearance 2.4-fold (p = 2 × 10 ). Minimal model analysis showed that exenatide increased glucose effectiveness (S ) by 32% (p = .0008) but did not significantly affect insulin sensitivity (S ). The exenatide-induced increase in insulin secretion made the largest contribution to interindividual variation in exenatide-induced acceleration of glucose disappearance while interindividual variation in the drug effect on S contributed to a lesser extent (β = 0.58 or 0.27, respectively).
CONCLUSIONS
This pilot study provides validation for the value of a frequently sampled intravenous glucose tolerance test (including minimal model analysis) to provide primary data for our ongoing pharmacogenomic study of pharmacodynamic effects of semaglutide (NCT05071898). Three endpoints provide quantitative assessments of the effects of glucagon-like peptide-1 receptor agonists on glucose metabolism: first phase insulin secretion, glucose disappearance rates and glucose effectiveness.
Topics: Humans; Exenatide; Diabetes Mellitus, Type 2; Glucose; Insulin Secretion; Hypoglycemic Agents; Glucagon-Like Peptide-1 Receptor; Pilot Projects; Glucagon-Like Peptide 1; Insulin; Peptides; Venoms; Blood Glucose
PubMed: 37264484
DOI: 10.1111/dom.15143 -
Nature Structural & Molecular Biology Dec 2023Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex...
Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. The mechanisms through which proteins are translocated via the TOM and TIM23 complexes remain unclear. Here we report the assembly of the active TOM-TIM23 supercomplex of Saccharomyces cerevisiae with translocating polypeptide substrates. Electron cryo-microscopy analyses reveal that the polypeptide substrates pass the TOM complex through the center of a Tom40 subunit, interacting with a glutamine-rich region. Structural and biochemical analyses show that the TIM23 complex contains a heterotrimer of the subunits Tim23, Tim17 and Mgr2. The polypeptide substrates are shielded from lipids by Mgr2 and Tim17, which creates a translocation pathway characterized by a negatively charged entrance and a central hydrophobic region. These findings reveal an unexpected pre-sequence pathway through the TOM-TIM23 supercomplex spanning the double membranes of mitochondria.
Topics: Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Carrier Proteins; Mitochondrial Membrane Transport Proteins; Saccharomyces cerevisiae Proteins; Protein Transport; Mitochondria; Saccharomyces cerevisiae; Mitochondrial Proteins; Peptides; Membrane Proteins
PubMed: 37696957
DOI: 10.1038/s41594-023-01103-7