ATPase Mutants Research Articles

Overexpressing the ClpC AAA+ unfoldase accelerates developmental cycle progression in Chlamydia trachomatis.

Chlamydia is an obligate intracellular bacterium that undergoes a complex biphasic developmental cycle, alternating between the smaller, infectious, non-dividing elementary body (EB) and the larger, non-infectious but dividing reticulate body. Due to the differences between these functionally and morphologically distinct forms, we hypothesize protein degradation is essential to chlamydial differentiation. The bacterial Clp system, consisting of an ATPase unfoldase (e.g., ClpX or ClpC) and a proteolytic component (e.g., ClpP), is critical for the physiology of bacteria through its recognition, and usually degradation, of specific substrates. We observed by transmission electron microscopy that overexpression of wild-type ClpC, but not an ATPase mutant isoform, in Chlamydia increased glycogen accumulation within the vacuolar niche of the bacteria earlier in the developmental cycle than typically observed. This suggested ClpC activity may increase the expression of EB-associated genes. Consistent with this, targeted RT-qPCR analyses demonstrated a significant increase in several EB-associated gene transcripts earlier in development. These effects were not observed with overexpression of the ATPase mutant of ClpC, providing strong evidence that the activity of ClpC drives secondary differentiation. By analyzing the global transcriptional response to ClpC overexpression using RNA sequencing, we observed a shift to earlier expression of canonical late developmental cycle genes and other EB-associated genes. Finally, we directly linked overexpression of ClpC with earlier production of infectious chlamydiae. Conversely, disrupting normal ClpC function with an ATPase mutant caused a delay in developmental cycle progression. Overall, these findings provide the first mechanistic insight for initiation of secondary differentiation in Chlamydia.IMPORTANCEChlamydia species are obligate intracellular bacteria that require a host cell in which to complete their unique developmental cycle. Chlamydia differentiates between an infectious but non-replicating form, the elementary body, and a non-infectious but replicating form, the reticulate body. The signals that drive differentiation events are not characterized. We hypothesize that proteases are essential for mediating differentiation by allowing remodeling of the proteome as the organism transitions from one functional form to another. We previously reported that the Caseinolytic protease (Clp) system is essential for chlamydial growth. Here, we reveal a surprising function for ClpC, an unfoldase, in driving production of infectious chlamydiae during the chlamydial developmental cycle.

Asymmetric Dynamics Drive Catalytic Activation of the Hsp90 Chaperone.

The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended 19F NMR spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to show that within the context of heterodimeric Hsp90, the conformationally dynamic subunit stimulates the ATPase activity of the normal subunit. The contrasting dynamic properties of the subunits within heterodimeric Hsp90 provide a mechanistic framework to understand the molecular basis for asymmetric Hsp90 activation and its importance for the biological function of Hsp90.

Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5898.

Cryo-EM structure and molecular dynamic simulations explain the enhanced stability and ATP activity of the pathological chaperonin mutant

Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the E.coli GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60V72I exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60V72I. Our structural analysis combined with molecular dynamic simulations showed mHsp60V72I with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60V72I mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant's ATPase activity. Our studies highlight the importance of mHsp60's characteristics in its biological function.

The Atpase Domain of LONP1 Is Necessary for Mitochondrial Protein Solubility and the Viability of Acute Myeloid Leukemia (AML) Cells

Compared to normal hematopoietic cells, we and others have shown that AML cells have a heightened reliance on oxidative phosphorylation and mitochondrial metabolism for survival and proliferation. To support this unique metabolic phenotype, we previously demonstrated that AML cells have increased import of nuclear-encoded mitochondrial proteins. These newly imported proteins must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins, and failure to properly process and fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on this family of mitochondrial chaperones, proteases, and heat shock proteins, we assessed their dependencies using the gene dependency datasets (eg: depmap.org). From this analysis, we identified the mitochondrial protease, LONP1, as the top hit, and among the top 10% of all essential genes for AML. Localized to the mitochondrial matrix, LONP1 is a nuclear encoded AAA+ serine protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to normal hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. Compared to normal hematopoietic cells (n=8) and CD34+ cells (n=3), LONP1 protein was increased in 16/30 primary AML samples by immunoblotting. Using shRNA and CRISPR, LONP1 knockdown and knockout reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX cells. Genetic knockdown or knockout of LONP1 increased levels of insoluble, aggregated mitochondrial proteins as measured by mass spectrometry, proteostat fluorescence and confocal microscopy, and immunoblotting of soluble and insoluble protein fractions. LONP1 knockdown/knockout also reduced mitochondrial respiration, depolarized the mitochondria, and induced AML cell death. We next determined the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival. We over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3'UTR of the endogenous gene. We then measured mitochondrial protein solubility/aggregation, mitochondrial respiration and cell viability. Wild type LONP1 and the proteolytically dead (S855A) mutant, but not the ATPase mutant (E591A), rescued mitochondrial protein solubility, mitochondrial respiration and cell viability, thus demonstrating that the ATPase domain of LONP1 is necessary for these functions. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that inhibits the ATPase activity of LONP1 by binding to an allosteric site near the ATPase domain of the enzyme. CDDO-Me killed OCI-AML2 and NB4 cells with IC 50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200nM) also killed >50% of cells in 3 out of 4 high-LONP1 expressing primary AML patient samples, but 10 of 10 tested primary AML samples with low levels of LONP1 were insensitive to the drug. Likewise, CDDO-Me induced mitochondrial protein aggregation in OCI-AML2 cells and in a primary AML patient sample with high LONP1 expression, while a primary AML patient sample with undetectable LONP1 expression showed no protein aggregation. In summary, the mitochondrial serine AAA+ protease LONP1 is over-expressed in a subset of AML cells and primary samples. The ATPase domain is necessary for LONP1's function in maintaining mitochondrial protein solubility. Selective inhibition of this domain leads to mitochondrial protein aggregation, impaired mitochondrial respiration and AML cell death. Thus, inhibiting the ATPase domain of LONP1 may be a novel therapeutic strategy for AML.

OR02-04 Peripheral Blood Steroid Profiling Discriminates Aldosterone-producing Adenoma Genotypes From Bilateral Primary Aldosteronism

Abstract Disclosure: A.F. Turcu: None. C. Lee: None. Z. Salman: None. H. Liu: None. A. Rabbah: None. S.M. Konzen: None. W.E. Rainey: None. T.J. Giordano: None. L. Zhao: None. A. Udager: None. Background: Primary aldosteronism (PA) is dichotomized into 2 major subtypes: unilaterally dominant PA, often caused by aldosterone-producing adenomas (APAs), and bilateral PA (BPA). Adrenal vein sampling (AVS)-guided unilateral adrenalectomy is often curative in patients with APA and offers marked cardiovascular and renal benefits. The diagnosis and subtyping of PA is a complex, multistep process, hindered by expertise and resource limitations. Objective: To develop steroid fingerprints that can distinguish genotype specific APAs from BPA in peripheral blood. Design And Methods: We enrolled 120 patients with PA who underwent AVS in a single tertiary referral center. Liquid-chromatography tandem mass spectrometry was used to simultaneously quantify 17 steroids in adrenal veins (AV) and periphery, both at baseline, and 10-20 min after cosyntropin stimulation. Aldosterone synthase (CYP11B2) immunohistochemistry-guided next generation sequencing was employed to identify aldosterone-driver mutations. Kruskal-Wallis tests were performed for comparison of continuous variables across multiple groups. Receiver operating characteristic (ROC) curves were plotted to discriminate APAs from BPA. Results: The 120 study participants included 75 patients with APA who underwent unilateral adrenalectomy and had documented aldosterone-driver mutations, and 45 patients with BPA. Of patients with APAs, 36 carried somatic mutations in CACNA1D, 24 in ATPase, and 15 in KCNJ5 genes, respectively. Patients with KCNJ5 mutations were younger and most were women. In the dominant AVs, the KCNJ5 group had marked elevations of 18-hydroxycortisol, 18-oxocortisol, aldosterone, and 11-deoxycorticosterone (DOC); conversely, the BB group had the lowest concentrations of mineralocorticoid and androgen precursors, despite comparable cortisol levels across all groups. Similar patterns were observed in peripheral blood. Linear discriminant analysis incorporating all steroids measured in baseline peripheral serum showed areas under the curve (AUC) of 0.98, 0.97, and 0.92 for distinguishing APAs with KCNJ5, ATPase, or CACNA1D mutations, respectively, from BPA. Conclusions: Single-assay steroid profiles measured in peripheral serum can distinguish genotype-specific APAs from BPA. Once validated, such tests might circumvent the need for AVS in large subset of PA patients. Presentation: Thursday, June 15, 2023

RecF protein targeting to post-replication (daughter strand) gaps II: RecF interaction with replisomes.

The bacterial RecF, RecO, and RecR proteins are an epistasis group involved in loading RecA protein into post-replication gaps. However, the targeting mechanism that brings these proteins to appropriate gaps is unclear. Here, we propose that targeting may involve a direct interaction between RecF and DnaN. In vivo, RecF is commonly found at the replication fork. Over-expression of RecF, but not RecO or a RecF ATPase mutant, is extremely toxic to cells. We provide evidence that the molecular basis of the toxicity lies in replisome destabilization. RecF over-expression leads to loss of genomic replisomes, increased recombination associated with post-replication gaps, increased plasmid loss, and SOS induction. Using three different methods, we document direct interactions of RecF with the DnaN β-clamp and DnaG primase that may underlie the replisome effects. In a single-molecule rolling-circle replication system in vitro, physiological levels of RecF protein trigger post-replication gap formation. We suggest that the RecF interactions, particularly with DnaN, reflect a functional link between post-replication gap creation and gap processing by RecA. RecF's varied interactions may begin to explain how the RecFOR system is targeted to rare lesion-containing post-replication gaps, avoiding the potentially deleterious RecA loading onto thousands of other gaps created during replication.

Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

Nanotheranostic: The futuristic therapy for copper mediated neurological sequelae

Copper (Cu) is contemplated as an indispensable metal with an exquisite role in nerve conduction, mitochondrial ATP synthesis, neurotransmitter synthesis and antioxidant activity. Cu imbalance is mainly prompted due to mutation in ATP7A and ATP7B ATPase transporting channels. The correlation of Cu disturbances and its impact on neurological sequelae was evaluated under various experiments. This correlation highlighted the role of Cu imbalance in the development of various neurological challenges namely, Alzheimer's disease, Parkinson's disease, Huntington's disease, Autism, Creutzfeldt-Jacob's disease and distal motor neuropathy. Although various Cu chelating agents along with theranostics are assessed in various clinical studies owing to its radionuclide capability. This review begins with the comprehension of Cu cerebral transportation, impact of Cu on progression of neurodegenerative disorder, followed by therapeutic approaches encompassing Cu as a nanotheranostic tool and nano chelators directed towards Cu chelation. Further nanotechnological platforms employed for nano-chelation were discussed. Moreover, we have discussed clinical status of copper as theranostic and chelating agents for copper as this will provide better idea of safety, tolerability and efficacy of these regimens. Overall chemo-sensitive therapies triggered with chemo-dynamic therapy, photothermal and other non-invasive imaging techniques can improve the theranostic efficacy. However, multifaceted nanocarriers can improve the chelation ability of nanocarriers. Harnessing these sophisticated methodologies can upgrade the copper chelation and theranostic safety and efficacy in clinical horizon.

Cohesin ATPase activities regulate DNA binding and coiled-coil configuration

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.

ACA pumps maintain leaf excitability during herbivore onslaught

Recurrent damage by lepidopteran folivores triggers repeated leaf-to-leaf electrical signaling. We found that the ability to propagate electrical signals-called slow wave potentials-was unexpectedly robust and was maintained in plants that had experienced severe damage. We sought genes that maintain tissue excitability during group insect attack. When Arabidopsis thaliana P-Type II Ca2+-ATPase mutants were mechanically wounded, all mutants tested displayed leaf-to-leaf electrical signals. However, when the auto-inhibited Ca2+-ATPase double-mutant aca10 aca12 was attacked by Spodoptera littoralis caterpillars, electrical signaling failed catastrophically, and the insects consumed these plants rapidly. The attacked double mutant displayed petiole base deformation and chlorosis, which spread acropetally into laminas and led to senescence. A phloem-feeding aphid recapitulated these effects, implicating the vasculature in electrical signaling failure. Consistent with this, ACA10 expressed in phloem companion cells in an aca10 aca12 background rescued electrical signaling and defense during protracted S.littoralis attack. When expressed in xylem contact cells, ACA10 partially rescued these phenotypes. Extending our analyses, we found that prolonged darkness also caused wound-response electrical signaling failure in aca10 aca12 mutants. Our results lead to a model in which the plant vasculature acts as a capacitor that discharges temporarily when leaves are subjected to energy-depleting stresses. Under these conditions, ACA10 and ACA12 function allows the restoration of vein cell membrane potentials. In the absence of these gene functions, vascular cell excitability can no longer be restored efficiently. Additionally, this work demonstrates that non-invasive electrophysiology is a powerful tool for probing early events underlying senescence.

Inhibition Plcγ2 and Hydrophobic Acids Synthesis cause Osteoarthritis, Diabetes and C-lymphocytic Leukemia Diseases Where Normally PLCs can Recover Interferons Synthesis

Proper S6K regulate BTK pathways which regulate PLCγ2 synthesis which are main regulations for thromboxane-A “TXA2” synthesis, and necessary for B-cell maturations and T-cells modulations and functions. Deficiency in Ser amino acids and in hydrophobic amino acids are reflect decreasing in synthase enzyme lead to deficiency in BTK function and deficiency in PLCγ2 that lead increasing in colony stimulating Factor-1 “CSF-1 where PLCγ1 specified to circuit to CSF-1 which upon synthase effect will promote PLCγ2 synthesis which necessary for activating BCRs for activating both IgM and IgD antigen for B-cells maturation and activities, for T-cells modulations, and for TXA2 synthesis. Proper Akt, S6K1 synthesis, OPA1 enzymes and fatty Acyl-COAs are necessary for regulating RORs isoforms Biosynthesis which regulate both IFNs and PLCs synthesis {Where both IFNs and PLCs are covering each other (IFNs <¬>PLCs) } that PLCγ1 promote the PLCγ2 synthesis upon BTK regulations. Osteoarthritis “OA” is characterized by a sharp expression in Gamma-Phospholipase C-1 “PLCγ1”, with decreasing “or inhibition” in PLCγ2 which reflect decreasing in synthase functions and in IFN-beta synthesis that reflect decreasing or deficiency in TXA2 Biosynthesis. The increasing in PLCγ1 with Deficiency in Ser amino acids will lead to deficiency in Ser phosphorylation signaling and deficiency in the pyrimidine kinases (PST-thymine and PS T-Cytosine kinases) synthesis, that lead to decreasing in synthase activity which will reflect down regulations in BTK pathways and inhibition in PLCγ2 productions which will reflect diabetes ( production of Androgen instead of estrogen), and can reflect Osteoarthritis “OA” prognosis which depend on the percentage of Deficiency or inhibition in basic amino acids and their basic necessary signaling pathways. T2DM is strongly connected with OA diseases and are linked together by the deficiency in Ser amino acids and their phosphorylation, and any early decreasing in Ser and in hydroponic acids synthesis can lead to both and more disease. Pathogenic type 2 diabetes associated with progressive beta-cell impairment due to the mutations in the production of S6K1 (deficiency in Ser ”TCT, TCC,TCA”), and inhibition in the PLCγ2 which due to inhibition or decreasing in Synthase and lead to deficiency in BCR activities. The decreasing in PS/T-Thymine Kinases and PS/T-Cytosine kinases chains (mTORC1) due to deficiency in Ser amino acids (where normally those pyrimidine kinases are produced from the phosphorylation process on Ser amino acids) will lead to mutated S6K and Akt productions and decreasing or mutations in ATPase and GTPase which lead to decreasing in OPA1 repair and lead to synthesis of Androgen instead of Estrogen which are depending on the availability of hydrophobic amino acids synthesis including Ser and Tyr amino acids. The effect of Synthetase enzymes on biological molecules is for creating active gamma-subunits “PLCγ1” that can be modified by synthase effect for Betasubunit synthesis “PLCγ2” then will be modified by phospholipase effects for alpha subunits productions. The releasing of pyrimidine kinases “PS/T-Thymine -Kinase and PS/T-Cytosine -kinase chains (mTORC1)” are so necessary steps for proper S6K productions and for proper fatty Acyl-COAs synthesis (long fatty AcylCOA) which are so important for regulating RORs Biosynthesis and necessary for both IFNs and for PLCs productions which started by the productions of PLC-gamma and IFN gamma, where both PLCγ1 and IFN-Gamma are necessary for regulating proper PLCγ2 biosynthesis upon “BTK activity” then PLCγ2 are necessary for regulating BCR functions which imp for regulating both IgM and IgD activities for B-cell maturations, for adjusting anti-inflammatory processes and for T-cells modulations, then PLCγ2 is so necessary for thromboxane-A synthesis, and for bone growth and immune modulations. Deficiency in conversion of glutarate to glutamate and decreasing in Proline (hydroponic acids) biosynthesis can affect on Cartilage synthesis and bone growth due to decreasing in stimulating mitochondrial OPA1 oxidations. Protein tyrosine phosphatase (PTP) gamma (carry−ve charge regulated firstly by synthetase gamma-oxidations) has been proposed to be an important regulator for chondrogenic patterning, where PTPs are critical regulators of tyrosine phosphorylation that it’s activity depends on the Tyr, and Ser synthesis (during hydrophobic acids synthesis ) and on JAK state signaling activities. And so, the proline-rich tyrosine kinases regulate proper PLCs isoforms which compete for binding site at the very C terminus of fibroblast growth factor for osteorogenitor embryonic development, and bone growth.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Asymmetric organelle positioning during epithelial polarization of C. elegans intestinal cells

While the epithelial cell cortex displays profound asymmetries in protein distribution and morphology along the apico-basal axis, the extent to which the cytoplasm is similarly polarized within epithelial cells remains relatively unexplored. We show that cytoplasmic organelles within C. elegans embryonic intestinal cells develop extensive apico-basal polarity at the time they establish cortical asymmetry. Nuclei and conventional endosomes, including early endosomes, late endosomes, and lysosomes, become polarized apically. Lysosome-related gut granules, yolk platelets, and lipid droplets become basally enriched. Removal of par-3 activity does not disrupt organelle positioning, indicating that cytoplasmic apico-basal asymmetry is independent of the PAR polarity pathway. Blocking the apical migration of nuclei leads to the apical positioning of gut granules and yolk platelets, whereas the asymmetric localization of conventional endosomes and lipid droplets is unaltered. This suggests that nuclear positioning organizes some, but not all, cytoplasmic asymmetries in this cell type. We show that gut granules become apically enriched when WHT-2 and WHT-7 function is disrupted, identifying a novel role for ABCG transporters in gut granule positioning during epithelial polarization. Analysis of WHT-2 and WHT-7 ATPase mutants is consistent with a WHT-2/WHT-7 heterodimer acting as a transporter in gut granule positioning. In wht-2(−) mutants, the polarized distribution of other organelles is not altered and gut granules do not take on characteristics of conventional endosomes that could have explained their apical mispositioning. During epithelial polarization wht-2(−) gut granules exhibit a loss of the Rab32/38 family member GLO-1 and ectopic expression of GLO-1 is sufficient to rescue the basal positioning of wht-2(−) and wht-7(−) gut granules. Furthermore, depletion of GLO-1 causes the mislocalization of the endolysosomal RAB-7 to gut granules and RAB-7 drives the apical mispositioning of gut granules when GLO-1, WHT-2, or WHT-7 function is disrupted. We suggest that ABC transporters residing on gut granules can regulate Rab dynamics to control organelle positioning during epithelial polarization.

Guard cell endomembrane Ca2+-ATPases underpin a 'carbon memory' of photosynthetic assimilation that impacts on water-use efficiency.

Stomata of most plants close to preserve water when the demand for CO2 by photosynthesis is reduced. Stomatal responses are slow compared with photosynthesis, and this kinetic difference erodes assimilation and water-use efficiency under fluctuating light. Despite a deep knowledge of guard cells that regulate the stoma, efforts to enhance stomatal kinetics are limited by our understanding of its control by foliar CO2. Guided by mechanistic modelling that incorporates foliar CO2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca2+ stores in guard cell responsiveness to fluctuating light and CO2. Modelling predicted and experiments demonstrated a delay in Ca2+ cycling that was enhanced by endomembrane Ca2+-ATPase mutants, altering stomatal conductance and reducing assimilation and water-use efficiency. Our findings illustrate the power of modelling to bridge the gap from the guard cell to whole-plant photosynthesis, and they demonstrate an unforeseen latency, or 'carbon memory', of guard cells that affects stomatal dynamics, photosynthesis and water-use efficiency.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.

Dynamic membranes: the multiple roles of P4 and P5 ATPases.

The lipid bilayer of biological membranes has a complex composition, including high chemical heterogeneity, the presence of nanodomains of specific lipids, and asymmetry with respect to lipid composition between the two membrane leaflets. In membrane trafficking, membrane vesicles constantly bud off from one membrane compartment and fuse with another, and both budding and fusion events have been proposed to require membrane lipid asymmetry. One mechanism for generating asymmetry in lipid bilayers involves the action of the P4 ATPase family of lipid flippases; these are biological pumps that use ATP as an energy source to flip lipids from one leaflet to the other. The model plant Arabidopsis (Arabidopsis thaliana) contains 12 P4 ATPases (AMINOPHOSPHOLIPID ATPASE1-12; ALA1-12), many of which are functionally redundant. Studies of P4 ATPase mutants have confirmed the essential physiological functions of these pumps and pleiotropic mutant phenotypes have been observed, as expected when genes required for basal cellular functions are disrupted. For instance, phenotypes associated with ala3 (dwarfism, pollen defects, sensitivity to pathogens and cold, and reduced polar cell growth) can be related to membrane trafficking problems. P5 ATPases are evolutionarily related to P4 ATPases, and may be the counterpart of P4 ATPases in the endoplasmic reticulum. The absence of P4 and P5 ATPases from prokaryotes and their ubiquitous presence in eukaryotes make these biological pumps a defining feature of eukaryotic cells. Here, we review recent advances in the field of plant P4 and P5 ATPases.

Astrocyte deletion of \u03b12-Na/K ATPase triggers episodic motor paralysis in mice via a metabolic pathway

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.