Dynamic structural profiling of PINK1 mutations (T313M and L347P) reveals a vital molecular perturbation namely phospho-Serine 65 ubiquitin recognition point in mitophagy mediated autosomal recessive Parkinson’s disease (ARPD)

Computational Characterization of Pathogenic LMNA Missense Variants: Structural Instability, Altered Binding, and Conformational Dynamics

Background Mutations in the LMNA gene underlie a broad spectrum of laminopathies, including muscular dystrophies, cardiomyopathies, and premature aging syndromes; however, the molecular mechanisms by which missense variants disrupt Lamin A structural integrity remain incompletely characterized. Systematic computational approaches for prioritizing pathogenic variants and elucidating their structural consequences are critically needed. Methods An integrated multistep in silico framework was employed to investigate the structural and functional consequences of LMNA missense variants. Variant prioritization was performed using the Evo2 nucleotide language model via delta log‐likelihood scoring, followed by bioinformatic annotation using SIFT, PANTHER‐PSEP, PhD‐SNP, and E‐SNPs&GO. Protein stability assessment was conducted with DynaMut, INPS‐MD, I‐Mutant2.0, and MUpro. Variants localized within globular domains—N456D, N456T, and G465D—together with the known pathogenic variant M540T as a positive control, were selected for three‐dimensional structural modeling using PyMOL and AlphaFold2, molecular docking with lonafarnib as a reference ligand via AutoDock Vina, and 100 ns molecular dynamics simulations using GROMACS with the Amber ff14SB force field. Conformational dynamics were characterized through principal component analysis and free‐energy surface construction. Results Evo2‐based screening of the full LMNA coding sequence identified 50 high‐priority loss‐of‐function variants, of which N456D, N456T, and G465D were retained for structural investigation based on their globular domain localization and multitool pathogenicity predictions. All three variants were consistently predicted to alter physicochemical properties and reduce structural stability relative to wild‐type Lamin A. Molecular docking revealed mutation‐dependent changes in lonafarnib binding profiles. The known pathogenic control M540T exhibited comparable structural and dynamic behavior, supporting the reliability of the prioritization workflow. Molecular dynamics analyses demonstrated altered RMSD trajectories, increased residue‐level flexibility, and modified hydrogen bonding patterns in mutant systems. Free‐energy landscape analyses revealed expanded conformational basins, particularly pronounced in the G465D variant, indicating increased structural plasticity. Conclusion This integrated computational framework provides a systematic strategy for prioritizing pathogenic LMNA variants and characterizing their structural consequences at the atomic level. The identified variants—N456D, N456T, and G465D—represent structurally disruptive substitutions consistent with the established role of globular domain destabilization in other laminopathy‐associated variants, offering testable hypotheses for experimental validation in cellular and animal models.

Effects of homozygous PARK7 gene mutations L166P and M26I on BNIP3/BNIP3L interactions and ER-mitochondria proximity

Mutations in PARK7, which encodes DJ-1, cause autosomal recessive early-onset Parkinson’s disease. DJ-1 contributes to mitochondrial homeostasis and ER-mitochondria communication, but how the pathogenic L166P and M26I variants affect BNIP3/BNIP3L-associated phenotypes remains incompletely understood. Here, we examined DJ-1 variant-dependent changes in BNIP3/BNIP3L interactions, mitochondrial morphology, ER-mitochondria proximity-associated readouts, and global intracellular Ca 2+ responses in PC-12 and SH-SY5Y cell models. AlphaFold3-based interface prediction, co-immunoprecipitation, and GST pull-down assays supported interactions involving DJ-1, BNIP3, and BNIP3L, including binding to selected BNIP3- and BNIP3L-derived peptide regions. DJ-1 WT and M26I showed detectable binding to BNIP3-derived regions, whereas the unstable L166P variant showed reduced BNIP3 binding even after partial restoration of L166P abundance with MG132. Reciprocal co-immunoprecipitation and knockdown experiments further supported an association between BNIP3 and BNIP3L in these cells. In L166P expressing cells with GRP75 knockdown, BNIP3 depletion increased the ER-mitochondria distance and reduced the length of the ER-mitochondria proximity region. Colocalization analyses showed DJ-1 variant-dependent changes in proximity-associated imaging readouts, while immunoblotting identified reduced VDAC1 and MFN1 levels after BNIP3 depletion in mutant expressing cells. BNIP3 depletion reduced the relative 2-APB evoked Fluo-4 response in WT, L166P and M26I expressing cells by 40.7%, 76.7%, and 42.5%, respectively. Overall, these findings may reflect altered DJ-1/BNIP3/BNIP3L interaction profiles and BNIP3 sensitive changes in ER-mitochondria proximity associated and global intracellular Ca 2+ readouts, particularly in L166P-expressing cells, although direct validation is still required.

LRRK2: Molecular Mechanisms in Parkinson’s Disease

Leucine-rich repeat kinase 2 (LRRK2) has emerged as a central molecular node linking genetic risk, membrane trafficking, lysosomal homeostasis, and immune signalling in Parkinson’s disease (PD). Rather than functioning as a conventional protein kinase, LRRK2 operates as a conformationally regulated, Rab-directed signalling machine whose activity is governed by long-range interdomain communication, membrane recruitment, and cooperative interactions with small GTPases. Converging advances in cryo-electron microscopy, quantitative phosphoproteomics, and human genetics indicate that pathogenic mutations, lysosomal stress, and pharmacological inhibitors do not simply alter catalytic output, but reshape the conformational landscape of LRRK2, biasing it toward distinct structural states with divergent cellular consequences. A defining feature of this system is the selective phosphorylation of Rab GTPases at low stoichiometry—most prominently Rab8 and Rab10—yet with disproportionate functional impact on vesicle trafficking, ciliogenesis, autophagy, and organelle positioning. The identification of Rab-directed phosphatases, particularly PPM1H, further establishes that LRRK2 signalling is governed by a dynamically balanced kinase–phosphatase circuit operating in space and time. These observations, together with emerging evidence linking LRRK2 activation to lysosomal damage and immune pathways, support a unifying hypothesis: PD-associated LRRK2 dysfunction arises from maladaptive stabilization of specific conformational and spatial states within a membrane-responsive signalling network, leading to persistent misregulation of Rab-dependent trafficking and organelle homeostasis, rather than from kinase hyperactivity alone. In this review, we integrate structural, biochemical, and cellular evidence to advance this framework and discuss its implications for disease mechanisms and therapy. We highlight key unresolved challenges—including conformation-selective drug targeting, spatial control of Rab phosphorylation, and context-dependent immune–neuronal crosstalk—and propose that restoring physiological regulation of LRRK2, rather than simply inhibiting its activity, will be essential for achieving mechanism-based disease modification in Parkinson’s disease.

MOLECULAR BASIS FOR PINK1 MATURATION

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson’s disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90alpha/cell division cycle 37/FK506-binding protein 51 (HSP90α/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography– tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine–proline–phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90α/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

Dissecting the transcriptome of Parkin (PRKN)-linked Parkinson’s disease using 2D and 3D hiPSC-neuronal models

This work generates human induced pluripotent stem cell-derived midbrain dopaminergic neurons using both a 2D differentiation protocol and a recently developed 3D method based on the microencapsulation of hiPSCs in small alginate/fibronectin beads.

PIK3CA in-frame deletion-activated PI3K/AKT signaling pathway drives macrodactyly.