Molecular and functional characterization of the retinitis pigmentosa G90V mutation in a conformationally stabilized rhodopsin background

International Journal of Biological Macromolecules · 2025-10-04

Mutations in the photoreceptor protein rhodopsin can lead to visual dysfunction and retinal degeneration. The G90 2.57 V mutation, in the second transmembrane helix, causes a retinitis pigmentosa phenotype. A conformationally stabilized wild-type rhodopsin bearing an engineered disulfide bond (N2C/D282C) was developed for structural studies of disease-associated mutations. However, this extra disulfide bond may mask the native conformational features of rhodopsin mutants. Here, we investigate the structural and functional consequences of the G90 2.57 V mutation in this stabilized context. The G90 2.57 V substitution disrupts interactions within the retinal binding pocket, particularly with E113 3.28 in transmembrane helix 3, which stabilizes the Schiff base linkage to 11- cis -retinal. Our results demonstrate that the N2C/D282C disulfide bond counteracts the destabilizing effects of the G90 2.57 V mutation by enhancing thermal and chemical stability of the pigment and improving chromophore regeneration. These findings underscore the importance helix and loop interactions in rhodopsin function and highlight the potential of structural modifications to rescue impaired mutants. Furthermore, our work provides novel insights into the effect of engineered disulfide bonds on the structure and dynamics of rhodopsin mutants associated with retinal diseases and allows one to dissect the effects of the disulfide bond from those of the rhodopsin mutation alone.

Aβ40 Fibril Assembly on Human Cerebral Smooth Muscle Cells Impairs Cell Viability

Biochemistry · 2025-01-07 · 2 citations

Cerebral vascular deposition of the amyloid-β (Aβ) peptide, a condition known as cerebral amyloid angiopathy (CAA), is associated with intracerebral hemorrhaging and contributes to disease progression in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID). Familial mutations at positions 22 and 23 within the Aβ peptide lead to early onset and severe CAA pathology. Here, we evaluate the effects of fibrillar Aβ peptides on the viability of primary-cultured human cerebral smooth muscle (HCSM) cells, which are the major site of amyloid deposition in cerebral blood vessel walls. Comparisons are made of the familial E22Q (Dutch) mutant of Aβ40 with wild-type Aβ40 and Aβ42. In agreement with previous studies, we find that there is a significant reduction in cell viability when Aβ40-Dutch or Aβ42-WT peptides are added to HCSM cell cultures as monomeric Aβ, whereas Aβ40-WT is relatively nontoxic. The binding of Aβ fibrils derived from sporadic CAA or familial Dutch-type CAA brain tissue to the membrane surface of HCSM cells does not result in a significant loss of cell viability. In contrast, when Aβ40-WT monomers and sporadic CAA fibrils are coincubated in HCSM cell cultures, there is a significant reduction in HCSM cell viability that is accompanied by an increase in cell surface fibril formation. Lastly, intrathecal administration of Aβ40-Dutch fibrillar seeds promotes fibrillar amyloid accumulation in the smooth muscle of meningeal vessels in the rTg-D transgenic rat model of CAA. Together, the present findings suggest that fibrillar Aβ seeds propagate the expansion of new amyloid fibrils on cerebral vascular smooth muscle, leading to membrane disruption and cell death.

An electrostatic cluster guides Aβ40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy

Journal of Structural Biology · 2024-04-12 · 18 citations

Author Correction: Cerebral vascular amyloid seeds drive amyloid β-protein fibril assembly with a distinct anti-parallel structure

Nature Communications · 2024-07-19

The original version of this Article contained an error in Fig. 4a and Fig. 4i, and in the figure legend for Fig. 4.In Fig. 4a, the representative image in this panel showing amyloid pathology in Tg-SwDI mice at 6 months of age was inadvertently duplicated from Fig. 4 of a previous publication by the same group 1 .In Fig. 4i, the representative image of capillaries with enlarged amyloid deposits that were observed in 18 months old bigenic Tg-SwDI/Tg2576 mice was an aggregate of representative amyloid laden vessels from three different brain regions used for the quantitative measures presented in panels (j-l) of Figure 4, but this was not demarcated on the figure panel.The representative vessels presented in the original composite image in Fig. 4i were not used for the quantitative comparisons of percentage A immune-positive capillaries (Fig. 4k) nor in measures of capillary amyloid volume (Fig. 4l) between the three brain regions.

Mechanism of Activation of the Visual Receptor Rhodopsin

Annual Review of Biophysics · 2023-05-09 · 23 citations

Rhodopsin is the photoreceptor in human rod cells responsible for dim-light vision. The visual receptors are part of the large superfamily of G protein-coupled receptors (GPCRs) that mediate signal transduction in response to diverse diffusible ligands. The high level of sequence conservation within the transmembrane helices of the visual receptors and the family A GPCRs has long been considered evidence for a common pathway for signal transduction. I review recent studies that reveal a comprehensive mechanism for how light absorption by the retinylidene chromophore drives rhodopsin activation and highlight those features of the mechanism that are conserved across the ligand-activated GPCRs.

Author response: Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers

2023-01-17

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers

eLife · 2023-06-20 · 8 citations

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Complete Genome Sequences of Actinobacteriophages Anaysia and Caviar

Microbiology Resource Announcements · 2022-10-26 · 1 citations

Anaysia and Caviar are temperate siphoviruses isolated from soil using Gordonia terrae 3612 and Mycobacterium smegmatis mc 2 155, respectively. Anaysia’s 52,861-bp genome carries 102 genes, while Caviar’s 47,074-bp genome carries 79 genes. Based on gene content similarity, Anaysia and Caviar are assigned to phage clusters A15 and A3, respectively.

Constitutive Activation and Oncogenicity Are Mediated by Loss of Helical Structure at the Cytosolic Boundary of the Thrombopoietin Receptor

bioRxiv (Cold Spring Harbor Laboratory) · 2022-06-30

Abstract Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with either proximity of Asn substitutions to the cytosolic boundary or mutation of W515 in the motif. Mutational studies in the TpoR cytosolic JM region show that the helical loss by itself can induce activation, but is localized to a maximum of 6 amino acids downstream of W515, the rest of the region until Box 1 requiring helicity for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation and to explain the critical role of the JM W515 residue in the regulation of receptor activity. (Total manuscript without References and Figure Legends 6507 words, main text (without methods and materials):)

An electrostatic cluster guides Aβ40 fibril formation in cerebral amyloid angiopathy

bioRxiv (Cold Spring Harbor Laboratory) · 2022-12-22 · 2 citations

ABSTRACT Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aβ peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer’s disease (AD). We investigate the structure of human-derived Aβ40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aβ40 fibril structures. One population has an ordered N-terminal fold comprised of two β-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations guide fibril formation.