Explore the vast range of titles available.

Abstract Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP–ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as β-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane–localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD–CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce “donut” mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1–CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1–CL interactions in stressinduced mitochondrial fission.

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington’s disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells. Huntington's disease is caused by a polyglutamine stretch expansion in the first exon of huntingtin. Here, the authors use infrared spectroscopy and solid-state NMR and show that polymorphic huntingtin exon1 fibres differ in their flanking regions but not their core polyglutamine amyloid structures.

Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils. Aggregation of eye lens proteins leads to cataracts, a major cause of blindness. Here the authors use solid state NMR to probe the structure of γD-crystallin eye lens proteins aggregates, which are found to retain a native-like conformation.

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1 . Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

In Huntington’s disease (HD), the polyglutamine (polyQ) expansion in huntingtin exon 1 (HttEx1) plays an important role in onset and progression of the disease. The age of onset (AO) is inversely dependent on the polyQ length beyond a threshold length near 35 residues. However, the molecular rationale behind this expansion threshold is still unclear. Understanding its mechanistic underpinnings of the AO may reveal ways to influence it, toward a much needed treatment of HD. We analyzed the aggregation behavior of HttEx1 proteins featuring polyQ segment lengths above and below the threshold (Q23-, Q32-, Q44- and Q80- HttEx1), to better understand threshold-related changes in fiber structure and mechanism of misfolding. We performed complementary techniques, including thioflavin T (ThT) assays, kinetic analysis, electron microscopy (EM) and state-of-the-art solid-state NMR. These analyses were applied to purified proteins (in vitro) and revealed differences in aggregation kinetics and fiber morphology. Yet, initial ssNMR analysis showed strong similarities for key aspects of the aggregated polyQ core that stabilizes the fibers. However, combining EM, ssNMR measurements and structural models of HttEx1 fibrils we identify a change in supramolecular arrangement of that polyQ segment as a hallmark of the below- vs above-threshold polyQ proteins. I will relate these findings to a mechanistic model (implicating beta-hairpin formation during nucleation) as a likely explanation of these findings. Altogether, these results shed light into how below and above the threshold polyQ length alters the mechanism of protein deposition process, not only in HD, but possibly also in other CAG expansion disorders.

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein 's expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of intermediate motion selection (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter's detection with specific dyes and positron emission tomography tracers. Competing Interest Statement The authors have declared no competing interest. Footnotes * The text was rewritten to obtain a better focus on the main findings. Some prior analysis (deconvolution and quantification) of several of the lower quality spectra was omitted as it was less reliable and detracted from the main message of the work. Several figures were updated with improved visualization and labeling. * https://doi.org/10.5281/zenodo.6552383

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of \"intrinsic\" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.