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PLoS Computational Biology May 2024The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress...
The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.
Topics: Kinesins; Protein Binding; Kinetics; Microtubules; Computational Biology; Adenosine Diphosphate; Computer Simulation; Models, Biological; Diffusion
PubMed: 38768214
DOI: 10.1371/journal.pcbi.1012158 -
Trials May 2024The SARS CoV-2 pandemic has resulted in more than 1.1 million deaths in the USA alone. Therapeutic options for critically ill patients with COVID-19 are limited. Prior...
SCARLET (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial): study protocol for a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 trial of i.v. citicoline (CDP-choline) in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory...
BACKGROUND
The SARS CoV-2 pandemic has resulted in more than 1.1 million deaths in the USA alone. Therapeutic options for critically ill patients with COVID-19 are limited. Prior studies showed that post-infection treatment of influenza A virus-infected mice with the liponucleotide CDP-choline, which is an essential precursor for de novo phosphatidylcholine synthesis, improved gas exchange and reduced pulmonary inflammation without altering viral replication. In unpublished studies, we found that treatment of SARS CoV-2-infected K18-hACE2-transgenic mice with CDP-choline prevented development of hypoxemia. We hypothesize that administration of citicoline (the pharmaceutical form of CDP-choline) will be safe in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory failure (HARF) and that we will obtain preliminary evidence of clinical benefit to support a larger Phase 3 trial using one or more citicoline doses.
METHODS
We will conduct a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 dose-ranging and safety study of Somazina® citicoline solution for injection in consented adults of any sex, gender, age, or ethnicity hospitalized for SARS CoV-2-associated HARF. The trial is named "SCARLET" (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial). We hypothesize that SCARLET will show that i.v. citicoline is safe at one or more of three doses (0.5, 2.5, or 5 mg/kg, every 12 h for 5 days) in hospitalized SARS CoV-2-infected patients with HARF (20 per dose) and provide preliminary evidence that i.v. citicoline improves pulmonary outcomes in this population. The primary efficacy outcome will be the SO:FO ratio on study day 3. Exploratory outcomes include Sequential Organ Failure Assessment (SOFA) scores, dead space ventilation index, and lung compliance. Citicoline effects on a panel of COVID-relevant lung and blood biomarkers will also be determined.
DISCUSSION
Citicoline has many characteristics that would be advantageous to any candidate COVID-19 therapeutic, including safety, low-cost, favorable chemical characteristics, and potentially pathogen-agnostic efficacy. Successful demonstration that citicoline is beneficial in severely ill patients with SARS CoV-2-induced HARF could transform management of severely ill COVID patients.
TRIAL REGISTRATION
The trial was registered at www.
CLINICALTRIALS
gov on 5/31/2023 (NCT05881135).
TRIAL STATUS
Currently enrolling.
Topics: Humans; Cytidine Diphosphate Choline; Double-Blind Method; SARS-CoV-2; COVID-19; Randomized Controlled Trials as Topic; COVID-19 Drug Treatment; Clinical Trials, Phase II as Topic; Pneumonia, Viral; Treatment Outcome; Hypoxia; Male; Pandemics; Coronavirus Infections; Hospitalization; Female; Betacoronavirus; Clinical Trials, Phase I as Topic; Respiratory Insufficiency; Administration, Intravenous; Adult
PubMed: 38760804
DOI: 10.1186/s13063-024-08155-0 -
Plant Molecular Biology May 2024Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine...
Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the K values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.
Topics: Synechocystis; Pyruvate Kinase; Phosphoenolpyruvate; Glucose-6-Phosphate; Ribosemonophosphates; Substrate Specificity; Hydrogen-Ion Concentration; Bacterial Proteins; Kinetics; Temperature
PubMed: 38758412
DOI: 10.1007/s11103-023-01401-0 -
Life Science Alliance Aug 2024Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis...
Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in , encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that in hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of .
Topics: Animals; Mice; Vinculin; Mice, Knockout; Muscular Dystrophies; Integrins; Choline Kinase; Sarcolemma; Humans; Focal Adhesions; Cell Membrane; Actinin; Muscle, Skeletal; Phosphatidylinositol 4,5-Diphosphate; Actins; Disease Models, Animal
PubMed: 38749543
DOI: 10.26508/lsa.202301956 -
Biochemical and Biophysical Research... Aug 2024Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible...
Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3'-OH of adenosine ribose' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.
Topics: Ubiquitin-Protein Ligases; Ubiquitination; Humans; Adenosine Diphosphate Ribose; Poly ADP Ribosylation; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Ubiquitin; ADP-Ribosylation; HEK293 Cells
PubMed: 38749191
DOI: 10.1016/j.bbrc.2024.150101 -
Biochemical and Biophysical Research... Jul 2024Cancer poses a significant risk to human well-being. Among the crucial characteristics of cancer is metabolic reprogramming. To meet the relentless metabolic needs,... (Review)
Review
Cancer poses a significant risk to human well-being. Among the crucial characteristics of cancer is metabolic reprogramming. To meet the relentless metabolic needs, cancer cells enhance cholesterol metabolism within the adverse tumor microenvironment. Reprograming cholesterol metabolism includes a series of modifications in the synthesis, absorption, esterification, and metabolites associated with cholesterol. These adjustments have a strong correlation with the proliferation, invasion, metastasis, and other characteristics of malignant tumors. FDFT1, also known as farnesyl diphosphate farnesyltransferase 1, is an enzyme crucial in the process of cholesterol biosynthesis. Its significant involvement in tumor metabolism has garnered considerable interest. The significance of FDFT1 in cancer metabolism cannot be overstated, as it actively interacts with cancer cells. This paper aims to analyze and consolidate the mechanism of FDFT1 in cancer metabolism and explore its clinical application. The goal is to contribute new strategies and targets for the prevention and treatment of cancer metabolism.
Topics: Humans; Neoplasms; Farnesyl-Diphosphate Farnesyltransferase; Cholesterol; Animals; Tumor Microenvironment
PubMed: 38749088
DOI: 10.1016/j.bbrc.2024.150046 -
BioRxiv : the Preprint Server For... May 2024Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins...
Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. Using parallel in vitro and in vivo CRISPR knockout screens, we identified terpenoid backbone biosynthesis genes as essential for pancreatic ductal adenocarcinoma (PDAC) tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that ( ) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model. These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.
PubMed: 38746286
DOI: 10.1101/2024.05.03.592368 -
BioRxiv : the Preprint Server For... May 2024In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal...
In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and β-farnesene.
PubMed: 38746203
DOI: 10.1101/2024.05.04.592530 -
Experimental and Clinical... Apr 2024Splenectomy during liver transplant can affect platelet function. In this study, our primary aim was to assess the perioperative platelet function by rotational...
Effect of Splenectomy on Coagulation and Platelet Function in Adult Liver Transplant Recipients Assessed With Rotational Thromboelastometry and Standard Coagulation Tests.
OBJECTIVES
Splenectomy during liver transplant can affect platelet function. In this study, our primary aim was to assess the perioperative platelet function by rotational thromboelastometry and the effects of splenectomy on platelet function.
MATERIALS AND METHODS
We studied 40 consecutive liver transplant recipients with end-stage liver disease (50% as a result of hepatitis C). Patients with splenectomy were compared with patients without splenectomy (n = 20/group). Three platelet function parameters by rotational thromboelastometry were studied: platelet activation with arachidonic acid, platelet activation with adenosine diphosphate, and platelet activation with thrombin receptor-activating peptide 6. Patients were monitored perioperatively and until postoperative day 21. Heparin was infused for 2 days postoperatively (60-180 U/kg/day), followed by administration of subcutaneous low-molecular-weight heparin (40 mg/24 h) on postoperative days 2 and 3 and oral acetylsalicylic acid when platelet count was >50 × 103/μL.
RESULTS
Liver disease contributed to low perioperative platelet count and function. Patients showed significant improvement by postoperative day 14 and day 21, particularly after splenectomy. Platelet count was significantly correlated with the 3 platelet function parameters by rotational thromboelastometry (P < .001). Acetyl salicylic acid was required earlier (postoperative day 3) for patients with splenectomy (8/20) but only affected the platelet function represented by platelet activation with arachidonic acid, whereas other platelet activation pathways were less affected. Patients received no transfusions of platelet units.
CONCLUSIONS
End-stage liver disease significantly contributed to low platelet function and counts before transplant. Two weeks were required for recovery of patients posttransplant, with further enhancement by splenectomy. Some recipients showed recovery that exceeded the normal reference range, which warranted monitoring. Acetyl salicylic acid only affected 1 platelet activation receptor.
Topics: Humans; Thrombelastography; Liver Transplantation; Male; Female; Middle Aged; Splenectomy; Treatment Outcome; Blood Coagulation; Adult; End Stage Liver Disease; Time Factors; Blood Platelets; Predictive Value of Tests; Platelet Activation; Platelet Function Tests; Platelet Aggregation Inhibitors; Anticoagulants; Platelet Count; Blood Coagulation Tests; Aspirin; Prospective Studies
PubMed: 38742319
DOI: 10.6002/ect.2023.0329 -
Frontiers in Plant Science 2024The uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products...
INTRODUCTION
The uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs).
METHODS
The genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis.
RESULTS
A total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements.
DISCUSSION
PgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.
PubMed: 38742211
DOI: 10.3389/fpls.2024.1363251