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Proceedings of the National Academy of... Jun 2024Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its...
Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.
Topics: Humans; Alcohol Dehydrogenase; Adipogenesis; Adipocytes; Tretinoin; Cell Differentiation; CRISPR-Cas Systems; Mutation, Missense; Adipose Tissue
PubMed: 38838011
DOI: 10.1073/pnas.2319301121 -
Frontiers in Endocrinology 2024A prothrombotic state was demonstrated in patients with Cushing's syndrome and is involved in the development and progression of cardiovascular and renal damage in...
BACKGROUND AND AIMS
A prothrombotic state was demonstrated in patients with Cushing's syndrome and is involved in the development and progression of cardiovascular and renal damage in hypertensive patients. This study was designed to examine the relationships between cortisol secretion and the hemostatic and fibrinolytic systems in hypertension.
METHODS
In 149 middle-aged, nondiabetic, essential hypertensive patients free of cardiovascular and renal complications, we measured hemostatic markers that express the spontaneous activation of the coagulation and fibrinolytic systems and assessed daily cortisol levels (8 AM, 3 PM, 12 AM; area under the curve, AUC-cortisol) together with the cortisol response to dexamethasone overnight suppression (DST-cortisol).
RESULTS
Plasma levels of D-dimer (D-dim), prothrombin fragment 1 + 2 (F1 + 2), and von Willebrand factor (vWF) were progressively and significantly higher across tertiles of AUC-cortisol and DST-cortisol, whereas no differences were observed in fibrinogen, tissue plasminogen activator, plasminogen activator inhibitor-1, antithrombin III, protein C, and protein S. D-dim, F1 + 2, and vWF were significantly and directly correlated with age and both AUC-cortisol and DST-cortisol. Multivariate regression analysis showed that both AUC-cortisol and DST-cortisol were related to plasma D-dim, F1 + 2, and vWF independently of age, body mass index, blood pressure, and renal function.
CONCLUSION
Greater daily cortisol profile and cortisol response to overnight suppression are independently associated with a prothrombotic state in hypertensive patients and might contribute to the development of organ damage and higher risk of cardiovascular complications.
Topics: Humans; Male; Middle Aged; Female; Hydrocortisone; Dexamethasone; Hypertension; Adult; Thrombosis; von Willebrand Factor; Circadian Rhythm; Aged; Biomarkers
PubMed: 38836224
DOI: 10.3389/fendo.2024.1397062 -
Cellular & Molecular Biology Letters Jun 2024The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given...
The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given autophagy functions to reduce surplus to compensate for scarcity, it theoretically possesses the capability to selectively degrade specific substrates to meet distinct metabolic demands. However, direct evidence is still lacking that substantiates the idea that autophagy selectively targets specific substrates (known as selective autophagy) to address particular nutritional needs. Recently, Gross et al. found that during phosphate starvation (P-S), rather than nitrogen starvation (N-S), yeasts selectively eliminate peroxisomes by dynamically altering the composition of the Atg1/ULK kinase complex (AKC) to adapt to P-S. This study elucidates how the metabolite sensor Pho81 flexibly interacts with AKC and guides selective autophagic clearance of peroxisomes during P-S, providing novel insights into the metabolic contribution of autophagy to special nutritional needs.
Topics: Autophagy; Phosphates; Saccharomyces cerevisiae Proteins; Peroxisomes; Saccharomyces cerevisiae; Autophagy-Related Protein-1 Homolog; Autophagy-Related Proteins; Protein Serine-Threonine Kinases; Protein Kinases
PubMed: 38834954
DOI: 10.1186/s11658-024-00597-3 -
Current Biology : CB Jun 2024The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the...
The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the kinetochore that integrates kinetochore attachment state with spindle checkpoint activity and kinetochore assembly.
Topics: Kinetochores; Chromosome Segregation; Microtubule-Associated Proteins; Cell Cycle Proteins; Saccharomyces cerevisiae Proteins; Spindle Apparatus; Protein Serine-Threonine Kinases; Microtubules; Saccharomyces cerevisiae
PubMed: 38834024
DOI: 10.1016/j.cub.2024.04.070 -
Nature Communications Jun 2024Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric...
Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.
Topics: Nuclear Envelope; Schizosaccharomyces; Telomere; Schizosaccharomyces pombe Proteins; Telomere-Binding Proteins; Anaphase; DNA Replication
PubMed: 38830842
DOI: 10.1038/s41467-024-48382-2 -
World Journal of Microbiology &... Jun 2024β-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we...
β-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve β-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for β-carotene production. Point and structural mutations were introduced into β-carotene-producing engineered yeast. The resulting mutants showed higher β-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of β-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and β-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with β-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.
Topics: Saccharomyces cerevisiae; beta Carotene; Metabolic Engineering; Saccharomyces cerevisiae Proteins; Mutation; Gene Expression Regulation, Fungal; Carotenoids; Mutagenesis; Point Mutation; Mevalonic Acid; Biosynthetic Pathways; Farnesyl-Diphosphate Farnesyltransferase
PubMed: 38829459
DOI: 10.1007/s11274-024-04037-4 -
MSystems Jun 2024Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions....
Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.
PubMed: 38829048
DOI: 10.1128/msystems.00305-24 -
PeerJ 2024The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s)...
BACKGROUND
The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum.
METHODS
The SAPs of clinical isolates of S. , , and were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM.
RESULTS
Immunoblot analysis revealed that sera from patients infected with . recognized 31 proteins. These SAP antigens are recognized by the host humoral response during infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in , and . This is the first report that demonstrates the presence of immunoreactive SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.
Topics: Humans; Feces; Dysentery, Bacillary; Shigella flexneri; Bacterial Proteins; Antibodies, Bacterial; Antigens, Bacterial; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Immunoglobulin A
PubMed: 38827305
DOI: 10.7717/peerj.17498 -
BioRxiv : the Preprint Server For... May 2024The risk of hypoglycemia and its serious medical sequelae restrict insulin replacement therapy for diabetes mellitus. Such adverse clinical impact has motivated...
UNLABELLED
The risk of hypoglycemia and its serious medical sequelae restrict insulin replacement therapy for diabetes mellitus. Such adverse clinical impact has motivated development of diverse glucose-responsive technologies, including algorithm-controlled insulin pumps linked to continuous glucose monitors ("closed-loop systems") and glucose-sensing ("smart") insulins. These technologies seek to optimize glycemic control while minimizing hypoglycemic risk. Here, we describe an alternative approach that exploits an endogenous glucose-dependent switch in hepatic physiology: preferential insulin signaling (under hyperglycemic conditions) preferential counter-regulatory glucagon signaling (during hypoglycemia). Motivated by prior reports of glucagon-insulin co-infusion, we designed and tested an ultra-stable glucagon-insulin fusion protein whose relative hormonal activities were calibrated by respective modifications; physical stability was concurrently augmented to facilitate formulation, enhance shelf life and expand access. An N-terminal glucagon moiety was stabilized by an α-helix-compatible Lys -Glu lactam bridge; A C-terminal insulin moiety was stabilized as a single chain with foreshortened C domain. Studies demonstrated (a) resistance to fibrillation on prolonged agitation at 37 °C and (b) dual hormonal signaling activities with appropriate balance. Glucodynamic responses were monitored in rats relative to control fusion proteins lacking one or the other hormonal activity, and continuous intravenous infusion emulated basal subcutaneous therapy. Whereas efficacy in mitigating hyperglycemia was unaffected by the glucagon moiety, the fusion protein enhanced endogenous glucose production under hypoglycemic conditions. Together, these findings provide proof of principle toward a basal glucose-responsive insulin biotechnology of striking simplicity. The fusion protein's augmented stability promises to circumvent the costly cold chain presently constraining global insulin access.
SIGNIFICANCE STATEMENT
The therapeutic goal of insulin replacement therapy in diabetes is normalization of blood-glucose concentration, which prevents or delays long-term complications. A critical barrier is posed by recurrent hypoglycemic events that results in short- and long-term morbidities. An innovative approach envisions co-injection of glucagon (a counter-regulatory hormone) to exploit a glycemia-dependent hepatic switch in relative hormone responsiveness. To provide an enabling technology, we describe an ultra-stable fusion protein containing insulin- and glucagon moieties. Proof of principle was obtained in rats. A single-chain insulin moiety provides glycemic control whereas a lactam-stabilized glucagon extension mitigates hypoglycemia. This dual-hormone fusion protein promises to provide a basal formulation with reduced risk of hypoglycemia. Resistance to fibrillation may circumvent the cold chain required for global access.
PubMed: 38826486
DOI: 10.1101/2024.05.20.594997 -
BioRxiv : the Preprint Server For... May 2024In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains...
In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the protein's C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.
PubMed: 38826232
DOI: 10.1101/2024.05.22.595309