Helicase Variants Assayed♦ : Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC2943293 | biostudies-literature | 2010 Sep
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.
| S-EPMC2943296 | biostudies-literature
Project description:The ring-shaped T7 helicase uses the energy of dTTP hydrolysis to perform the mechanical work of translocation and base pair (bp) separation. We have shown that the unwinding rate of T7 helicase decreases with increasing DNA stability. Here, we show that the dTTPase rate also decreases with increasing DNA stability, which indicates close linkage between chemical transition steps and translocation steps of unwinding. We find that the force-producing step during unwinding is not associated with dTTP binding, but dTTP hydrolysis or P(i) release. We determine that T7 helicase extracts approximately 3.7 kcal/mol energy from dTTPase to carry out the work of strand separation. Using this energy, T7 helicase unwinds approximately 4 bp of AT-rich DNA or 1-2 bp of GC-rich DNA. T7 helicase therefore adjusts both its speed and coupling ratio (bp/dTTP) to match the work of DNA unwinding. We discuss the mechanistic implications of the variable bp/dTTP that indicates T7 helicase either undergoes backward movements/futile hydrolysis or unwinds DNA with a variable bp-step size; 'long and fast' steps on AT-rich and 'short and slow' steps on GC-rich DNA.
| S-EPMC2435122 | biostudies-literature
Project description:The melanocortin 1 receptor (MC1R) is a highly polymorphic G protein-coupled receptor, which is known to modulate pigmentation and inflammation. In the current study, we investigated the pharmacological effects of select single-nucleotide polymorphisms (SNPs) (V60L, R163Q, and F196L). After transient expression of MC1Rs in human embryonic kidney 293 cells, basal and ligand-induced cAMP signaling and mitogen-activated protein kinase (MAPK) activation were assessed by using luciferase reporter gene assays and Western blot analysis, respectively. All receptor variants showed decreased basal cAMP activity. With the V60L and F196L variants, the decrease in constitutive activity was attributable, at least in part, to a reduction in surface expression. The F196L variant also displayed a significant reduction in potency for both the peptide agonist α-melanocyte-stimulating hormone (α-MSH) and the small-molecule agonist 1-[1-(3-methyl-L-histidyl-O-methyl-D-tyrosyl)-4-phenyl-4-piperidinyl]-1-butanone (BMS-470539). In MAPK signaling assays, the F196L variant showed decreased phospho-extracellular signal-regulated kinase levels after stimulation with either α-MSH or BMS-470539. In contrast, the R163Q variant displayed a selective loss of α-MSH-induced MAPK activation; whereas responsiveness to the small-molecule agonist BMS-470539 was preserved. Further assessment of MC1R variants in A549 cells, an in vitro model of inflammation, revealed an enhanced inflammatory response resulting from expression of the F196L variant (versus the wild-type MC1R). This alteration in function was restored by treatment with BMS-470539. Overall, these studies illustrate novel signaling profiles linked to distinct MC1R SNPs. Furthermore, our investigations highlight the potential for small-molecule drugs to rescue the function of MC1R variants that show reduced basal and/or α-MSH stimulated activity.
| S-EPMC3400803 | biostudies-literature
Project description:Amyloid-beta (Aβ) deposition as senile plaques is a pathological hallmark of Alzheimer's disease (AD). AD is characterized by a large level of heterogeneity in amyloid pathology, whose molecular origin is poorly understood. Here, we employ NMR spectroscopy and MD simulation at ambient and high pressures and investigate how AD-related mutations in Aβ peptide influence the stability of Aβ aggregates. The pressure-induced monomer dissociation from Aβ aggregates monitored by NMR demonstrated that the Iowa (D23N), Arctic (E22G), and Osaka (ΔE22) mutations altered the pressure stability of Aβ40 aggregates in distinct manners. While the NMR data of monomeric Aβ40 showed only small localized effects of mutations, the MD simulation of mutated Aβ fibrils revealed their distinct susceptibility to elevated pressure. Our data propose a structural basis for the distinct stability of various Aβ fibrils and highlights "stability" as a molecular property potentially contributing to the large heterogeneity of amyloid pathology in AD.
| S-EPMC9940190 | biostudies-literature
Project description:Defects in organelle dynamics underlie a number of human degenerative disorders, and whole exome sequencing (WES) is a powerful tool for studying genetic changes that affect the cellular machinery. WES may uncover variants of unknown significance (VUS) that require functional validation. Previously, a pathogenic de novo variant in the middle domain of DNM1L (p.A395D) was identified in a single patient with a lethal defect of mitochondrial and peroxisomal fission. We identified two additional patients with infantile encephalopathy and partially overlapping clinical features, each with a novel VUS in the middle domain of DNM1L (p.G350R and p.E379K). To evaluate pathogenicity, we generated transgenic Drosophila expressing wild-type or variant DNM1L. We find that human wild-type DNM1L rescues the lethality as well as specific phenotypes associated with the loss of Drp1 in Drosophila. Neither the p.A395D variant nor the novel variant p.G350R rescue lethality or other phenotypes. Moreover, overexpression of p.A395D and p.G350R in Drosophila neurons, salivary gland and muscle strikingly altered peroxisomal and mitochondrial morphology. In contrast, the other novel variant (p.E379K) rescued lethality and did not affect organelle morphology, although it was associated with a subtle mitochondrial trafficking defect in an in vivo assay. Interestingly, the patient with the p.E379K variant also has a de novo VUS in pyruvate dehydrogenase 1 (PDHA1) affecting the same amino acid (G150) as another case of PDHA1 deficiency suggesting the PDHA1 variant may be pathogenic. In summary, detailed clinical evaluation and WES with functional studies in Drosophila can distinguish different functional consequences of newly-described DNM1L alleles.
| S-EPMC5007591 | biostudies-literature
Project description:Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.
| S-EPMC4924935 | biostudies-literature
Project description:The DNA helicase encoded by gene 4 of bacteriophage T7 forms a hexameric ring in the presence of dTTP, allowing it to bind DNA in its central core. The oligomerization also creates nucleotide-binding sites located at the interfaces of the subunits. DNA binding stimulates the hydrolysis of dTTP but the mechanism for this two-step control is not clear. We have identified a glutamate switch, analogous to the glutamate switch found in AAA+ enzymes that couples dTTP hydrolysis to DNA binding. A crystal structure of T7 helicase shows that a glutamate residue (Glu-343), located at the subunit interface, is positioned to catalyze a nucleophilic attack on the γ-phosphate of a bound nucleoside 5'-triphosphate. However, in the absence of a nucleotide, Glu-343 changes orientation, interacting with Arg-493 on the adjacent subunit. This interaction interrupts the interaction of Arg-493 with Asn-468 of the central β-hairpin, which in turn disrupts DNA binding. When Glu-343 is replaced with glutamine the altered helicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. When both Arg-493 and Asn-468 are replaced with alanine, dTTP hydrolysis is no longer stimulated in the presence of DNA. Taken together, these results suggest that the orientation of Glu-343 plays a key role in coupling nucleotide hydrolysis to the binding of DNA.
| S-EPMC3123078 | biostudies-literature
Project description:Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5' terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5' terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5' terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3' terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5' terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.
| S-EPMC5067871 | biostudies-literature
Project description:By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here, we focus on dormancy in the Bacillota, where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship or reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. We characterized the distribution of sigma factors in over 4,000 genomes of diverse phages capable of infecting hosts that span the bacterial domain. From this, we identified homologs of sporulation-specific sigma factors in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, we provide evidence that sporulation-like sigma factors are nonessential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis, some of these phage-derived sigma factors can activate the bacterial sporulation gene network and lead to a reduction in spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth. IMPORTANCE As obligate parasites, phages exert strong top-down pressure on host populations with eco-evolutionary implications for community dynamics and ecosystem functioning. The process of phage infection, however, is constrained by bottom-up processes that influence the energetic and nutritional status of susceptible hosts. Many phages have acquired auxiliary genes from bacteria, which can be used to exploit host metabolism with consequences for phage fitness. In this study, we demonstrate that phages infecting spore-forming bacteria carry homologs of sigma factors, which their hosts use to orchestrate gene expression during spore development. By tapping into regulatory gene networks, phages may manipulate the physiology and survival strategies of nongrowing bacteria in ways that influence host-parasite coevolution.
| S-EPMC9429907 | biostudies-literature
Project description:Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.
| S-EPMC7914560 | biostudies-literature