Nitrogenase Complexes: Multiple Docking Sites for a Nucleotide Switch Protein

Adenosine triphosphate (ATP) hydrolysis in the nitrogenase complex controls the cycle of association and dissociation between the electron donor adenosine triphosphatase (ATPase) (Fe-protein) and its target catalytic protein (MoFe-protein), driving the reduction of dinitrogen into ammonia. Crystal structures in different nucleotide states have been determined that identify conformational changes in the nitrogenase complex during ATP turnover. These structures reveal distinct and mutually exclusive interaction sites on the MoFe-protein surface that are selectively populated, depending on the Fe-protein nucleotide state. A consequence of these different docking geometries is that the distance between redox cofactors, a critical determinant of the intermolecular electron transfer rate, is coupled to the nucleotide state. More generally, stabilization of distinct docking geometries by different nucleotide states, as seen for nitrogenase, could enable nucleotide hydrolysis to drive the relative motion of protein partners in molecular motors and other systems

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin’s stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration

Retinal Gene Distribution and Functionality Implicated in Inherited Retinal Degenerations Can Reveal Disease-Relevant Pathways for Pharmacologic Intervention

The advent of genetic therapies for inherited retinal diseases (IRDs) has spurred the need for precise diagnosis and understanding of pathways for therapeutic targeting. The majority of IRDs that are clinically diagnosed, however, lack an identifiable mutation in established disease-causing loci and thus can be investigated with limited rational drug discovery methods. Transcriptome profiling of the retina can reveal the functional state of the tissue, and geographic profiling can uncover the various clinical phenotypic presentations of IRDs and aid in pharmaceutical intervention. In this investigation, we detail the retinal geographic expression of known retinal disease-causing genes in the primate retina and functional targetable pathways in specific IRDs. Understanding the genetic basis as well as the resulting functional consequences will assist in the discovery of future therapeutic interventions and provide novel insights to medicinal chemists. Herein, we report that, despite the genetic heterogeneity of retinal diseases, potential functional pathways can be elucidated for therapeutic targeting and be used for predictive phenotypic and genotypic modeling of novel IRD presentations

Retinal Gene Distribution and Functionality Implicated in Inherited Retinal Degenerations Can Reveal Disease-Relevant Pathways for Pharmacologic Intervention

The advent of genetic therapies for inherited retinal diseases (IRDs) has spurred the need for precise diagnosis and understanding of pathways for therapeutic targeting. The majority of IRDs that are clinically diagnosed, however, lack an identifiable mutation in established disease-causing loci and thus can be investigated with limited rational drug discovery methods. Transcriptome profiling of the retina can reveal the functional state of the tissue, and geographic profiling can uncover the various clinical phenotypic presentations of IRDs and aid in pharmaceutical intervention. In this investigation, we detail the retinal geographic expression of known retinal disease-causing genes in the primate retina and functional targetable pathways in specific IRDs. Understanding the genetic basis as well as the resulting functional consequences will assist in the discovery of future therapeutic interventions and provide novel insights to medicinal chemists. Herein, we report that, despite the genetic heterogeneity of retinal diseases, potential functional pathways can be elucidated for therapeutic targeting and be used for predictive phenotypic and genotypic modeling of novel IRD presentations

Retinal Gene Distribution and Functionality Implicated in Inherited Retinal Degenerations Can Reveal Disease-Relevant Pathways for Pharmacologic Intervention.

The advent of genetic therapies for inherited retinal diseases (IRDs) has spurred the need for precise diagnosis and understanding of pathways for therapeutic targeting. The majority of IRDs that are clinically diagnosed, however, lack an identifiable mutation in established disease-causing loci and thus can be investigated with limited rational drug discovery methods. Transcriptome profiling of the retina can reveal the functional state of the tissue, and geographic profiling can uncover the various clinical phenotypic presentations of IRDs and aid in pharmaceutical intervention. In this investigation, we detail the retinal geographic expression of known retinal disease-causing genes in the primate retina and functional targetable pathways in specific IRDs. Understanding the genetic basis as well as the resulting functional consequences will assist in the discovery of future therapeutic interventions and provide novel insights to medicinal chemists. Herein, we report that, despite the genetic heterogeneity of retinal diseases, potential functional pathways can be elucidated for therapeutic targeting and be used for predictive phenotypic and genotypic modeling of novel IRD presentations

Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients

Abstract Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients with autosomal recessive IRDs, one or no disease-causing variants are identified. Furthermore, chromosomal maps cannot be reconstructed for allelic variant discovery with short-reads. Long-read genome sequencing can provide complete coverage of disease loci and a targeted approach can focus sequencing bandwidth to a genomic region of interest to provide increased depth and haplotype reconstruction to uncover cases of missing heritability. We demonstrate that targeted adaptive long-read sequencing on the Oxford Nanopore Technologies (ONT) platform of the USH2A gene from three probands in a family with the most common cause of the syndromic IRD, Usher Syndrome, resulted in greater than 12-fold target gene sequencing enrichment on average. This focused depth of sequencing allowed for haplotype reconstruction and phased variant identification. We further show that variants obtained from the haplotype-aware genotyping pipeline can be heuristically ranked to focus on potential pathogenic candidates without a priori knowledge of the disease-causing variants. Moreover, consideration of the variants unique to targeted long-read sequencing that are not covered by short-read technology demonstrated higher precision and F1 scores for variant discovery by long-read sequencing. This work establishes that targeted adaptive long-read sequencing can generate targeted, chromosome-phased data sets for identification of coding and non-coding disease-causing alleles in IRDs and can be applicable to other Mendelian diseases

R9AP Overexpression Alters Phototransduction Kinetics in iCre75 Mice

PURPOSE. Determine the impact of rod photoreceptor-specific expression of Cre recombinase on the kinetics of phototransduction in the mouse eye and identify changes in gene expression that underlie any observed phenotypic differences. METHODS. Transretinal ERG and single-cell suction electrode recordings were used to measure the kinetics of phototransduction in a mouse line exhibiting rod photoreceptor–specific Cre recombinase expression, and the results were compared with those from control non–Cre-expressing littermates. Gene expression changes were evaluated using RNA sequencing transcriptome analysis. The pattern of expression of Rgs9bp was determined by mapping sequencing reads to the mouse genome and performing 3′-rapid amplification of cDNA ends (3′-RACE). RESULTS. Expression of the rod-specific iCre75 transgene was accompanied by accelerated phototransduction inactivation, likely due to overexpression of the Rgs9bp gene, which encodes the Rgs9 anchor protein (R9AP). R9AP upregulation stabilized the RGS9 GAP complex, altering phototransduction kinetics. 3′-Race identified an abundant, unexpected Rgs9bp-Prm1 fusion mRNA in Cre-expressing mouse retinas, which was determined to be derived from a second transgene present in the iCre75 line. CONCLUSIONS. Here we report the presence of a second, R9AP-expressing transgene in the iCre75 mouse line, leading to altered kinetics of phototransduction. These results highlight an important caveat that must be considered when utilizing this mouse line for rod photoreceptor–specific gene loss of function studies