Calcium-Dependent RTX Domains in the Development of Protein Hydrogels.
Ontology highlight
ABSTRACT:
SUBMITTER: Bulutoglu B
PROVIDER: S-EPMC6473919 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms.
| S-EPMC6891781 | biostudies-literature
Project description:Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we use protein-polymer interactions to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as polyethyleneimine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels with tunable wide-range stiffness, from ~10-64 kPa, without affecting the protein mechanics and nanostructure. We use the 6-fold increase in stiffness induced by PEI to program BSA hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. The approach demonstrated here, based on protein engineering and polymer reinforcing, may enable the development and investigation of smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.
| S-EPMC6884551 | biostudies-literature
Project description:RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity between adhesins. Here we have produced and structurally characterized protein constructs of four tandem repeats (tetra-tandemers) from two different RTX adhesins. In comparing the tetra-tandemers to each other and already solved structures from Marinomonas primoryensis and Salmonella enterica, the extender domains fold as diverse beta-sandwich structures with widely differing calcium contents. However, all the tetra-tandemers have at least one calcium ion coordinated in the linker region between beta-sandwich domains whose role appears to be the rigidification of the extender region to help the adhesin extend its reach.
| S-EPMC7493085 | biostudies-literature
Project description:Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by so-called RTX (repeat-in-toxin) motifs found in many virulence factors secreted by numerous gram-negative pathogenic bacteria: RTX proteins are natively disordered in the absence of calcium but fold upon calcium binding. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, contains ∼40 RTX motifs organized in five successive blocks separated by non-RTX flanking regions. This RTX domain mediates toxin binding to its eukaryotic cell receptor. We previously showed that the last block of the RTX domain, block V, which is critical for CyaA toxicity, exhibits the hallmarks of intrinsically disordered proteins in the absence of calcium. Moreover, the C-terminal flanking region of CyaA block V is required for its calcium-induced folding. Here, we describe a comprehensive analysis of the hydrodynamic and electrophoretic properties of several block V RTX polypeptides that differ in the presence and/or length of the flanking regions. Our results indicate that the length of the C-terminal flanking region not only controls the calcium-induced folding but also the calcium-induced multimerization of the RTX polypeptides. Moreover, we showed that calcium binding is accompanied by a strong reduction of the net charge of the RTX polypeptides. These data indicate that the disorder-to-order transition in RTX proteins is controlled by a calcium-induced change of the polypeptide charges and stabilized by multimerization.
| S-EPMC3089544 | biostudies-literature
Project description:Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1(fl/fl) mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-Cre(ERT);Hes1(fl/fl) mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.
| S-EPMC4364241 | biostudies-literature
Project description:Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.
| S-EPMC8126791 | biostudies-literature
Project description:Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3' and 5'UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression.
| S-EPMC3414820 | biostudies-literature
Project description:Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated in part by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.
| S-EPMC3306180 | biostudies-literature
Project description:Elevation of intracellular calcium was traditionally thought to be detrimental in stroke pathology. However, clinical trials testing treatments that block calcium signaling have failed to improve outcomes in ischemic stroke. Emerging data suggest that calcium may also trigger endogenous protective pathways after stroke. Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is a major kinase activated by rising intracellular calcium. Compelling evidence has suggested that CaMKK and its downstream kinase CaMK IV are critical in neuronal survival when cells are under ischemic stress. We examined the functional role of CaMKK/CaMK IV signaling in stroke.We used a middle cerebral artery occlusion model in mice.Our data demonstrated that pharmacological and genetic inhibition of CaMKK aggravated stroke injury. Additionally, deletion of CaMKK ?, one of the 2 CaMKK isoforms, reduced CaMK IV activation, and CaMK IV deletion in mice worsened stroke outcome. Finally, CaMKK ? or CaMK IV knockout mice had exacerbated blood-brain barrier disruption evidenced by increased hemorrhagic transformation and activation of matrix metalloproteinase. We observed transcriptional inactivation including reduced levels of histone deacetylase 4 phosphorylation in mice with CaMKK ? or CaMK IV deletion after stroke.Our data have established that the CaMKK/CaMK IV pathway is a key endogenous protective mechanism in ischemia. Our results suggest that this pathway serves as an important regulator of blood-brain barrier integrity and transcriptional activation of neuroprotective molecules in stroke.
| S-EPMC3845442 | biostudies-literature
Project description:Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.
| S-EPMC6336795 | biostudies-literature