ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Loss-of-function mutations in ATP13A2 (PARK9) are causative for a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome (KRS, a juvenile onset parkinsonism with dementia) (1), early-onset Parkinson’s disease (PD) (2, 3), hereditary spastic paraplegia (HSP) (4), neuronal ceroid lipofuscinosis (5), and amyotrophic lateral sclerosis (6), which are commonly hallmarked by lysosomal and mitochondrial dysfunction (4, 6, 7). Also, ATP13A2 deficiency causes lysosomal and mitochondrial impairment in various models, as evidenced by decreased lysosomal functionality (8, 9), reduced mitochondrial clearance capacity (8–10), mitochondrial fragmentation, mitochondrial DNA damage, and increased oxygen consumption (11, 12).We recently discovered that ATP13A2 transports the polyamines spermidine and spermine from the late endo/lysosome to the cytosol (9). Polyamines are ubiquitous polycationic aliphatic amines that stabilize nucleic acids, influence protein folding, regulate ion channels, and modulate cell proliferation and differentiation (13–15). We found that the late endo-lysosomal transporter ATP13A2 strongly contributes to the total cellular polyamine content via a two-step process: Firstly, polyamines enter the cell via endocytosis and subsequently, polyamines are transported by ATP13A2 into the cytosol (9). This process complements polyamine biosynthesis via the ornithine decarboxylase (ODC) pathway (9). Importantly, ATP13A2’s polyamine transport function is crucial for its neuroprotective effect, since it prevents lysosomal polyamine accumulation and subsequent lysosomal rupture, while improving lysosomal health and functionality (9). Moreover, when activated by its two regulatory lipids—phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] and phosphatidic acid (PA)—ATP13A2 exerts a cell protective effect against the mitochondrial neurotoxin rotenone (16), an environmental risk factor for PD (17). Rotenone is a mitochondrial complex I inhibitor, which leads to high levels of reactive oxygen species (ROS), promoting protein aggregation and damaging organelles. However, how ATP13A2’s polyamine transport function exerts a cell protective effect against rotenone, or other mitochondrial neurotoxins, is not yet clear.Interestingly, the transported substrates spermine and spermidine reduce oxidative stress (14, 15). Spermine is a potent free radical scavenger (18) and a biologically important antioxidant (19–23). We therefore hypothesize that ATP13A2-mediated polyamine transport may counteract oxidative stress (16, 24) and preserve mitochondrial health (11, 12). Here, we demonstrate in complementary human cell models and Caenorhabditis elegans that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders.     

Distribution of light chains and ATPase activity of myosin in the atrioventricular conducting tissue of bovine heart

Summary The relations of the light chains of myosins of the atria, ventricles, and atrioventricular conducting tissue (specialized myocardial tissue) and the distribution of the light chains of myosin in different regions of the atrioventricular conducting tissue in bovine heart were examined. Two-dimensional gel electrophoresis showed that the atrial and ventricular myosins each had two light chains (LC₁ and LC₂). Ventricular LC₁ differed from atrial LC₁, but ventricular LC₂ corresponded to atrial LC₂. The specialized myocardial tissue myosin had three light chains (named here SL₁, SL₂, and SL₃). SL₁ comigrated with ventricular LC₁, SL₂ with atrial LC₁, and SL₃ with ventricular LC₂ and atrial LC₂. The compositions of the three light chains of myosins in various regions of the atrioventricular conducting tissue were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The percentage proportion of SL₁ decreased in the order—atrioventricular node (AVN), right and left bundle branches (RLBB), His bundle (HIS), false tendon (FT) myosin; while the percentage proportion of SL₂ decreased in the order—FT and HIS, RLBB, AVN myosin. The percentages of SL₃ in these four regions were similar. The Ca²⁺-activated ATPase activity of myosin was highest in the AVN and lowest in the FT. The activities in the HIS and RLBB were intermediate between those in the AVN and FT. Thus, the composition of the light chains and the Ca²⁺-activated ATPase activity were different in various regions of the atrioventricular conducting tissue.     

TCIRG1‐Associated Congenital Neutropenia

Severe congenital neutropenia (SCN) is a rare hematopoietic disorder, with estimated incidence of 1 in 200,000 individuals of European descent, many cases of which are inherited in an autosomal dominant pattern. Despite the fact that several causal genes have been identified, the genetic basis for >30% of cases remains unknown. We report a five‐generation family segregating a novel single nucleotide variant (SNV) in TCIRG1. There is perfect cosegregation of the SNV with congenital neutropenia in this family; all 11 affected, but none of the unaffected, individuals carry this novel SNV. Western blot analysis show reduced levels of TCIRG1 protein in affected individuals, compared to healthy controls. Two unrelated patients with SCN, identified by independent investigators, are heterozygous for different, rare, highly conserved, coding variants in TCIRG1.     

Disruption of copper homeostasis due to a mutation of Atp7a delays the onset of prion disease

Copper influences the pathogenesis of prion disease, but whether it is beneficial or detrimental remains controversial. Copper homeostasis is also essential for normal physiology, as highlighted by the spectrum of diseases caused by disruption of the copper transporting enzymes ATP7A and ATP7B. Here, by using a forward genetics approach in mice, we describe the isolation of three alleles of Atp7a, each with different phenotypic consequences. The mildest of the three, Atp7a(brown), was insufficient to cause lethality in hemizygotes or mottling of the coat in heterozygotes, but did lead to coat hypopigmentation and reduced copper content in the brains of hemizygous males. When challenged with Rocky Mountain Laboratory scrapie, the onset of prion disease was delayed in Atp7a(brown) mice, and significantly less proteinase-resistant prion protein was found in the brains of moribund Atp7a(brown) mice compared with WT littermates. Our results establish that ATP7A-mediated copper homeostasis is important for the formation of pathogenic proteinase-resistant prion protein.     

Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy

p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.     

Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations

The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.     

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.     

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.     

Structure–Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97

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