Functional and in silico analysis of ATP8A2 and other P4-ATPase variants associated with human genetic diseases.

P4-ATPases flip lipids from the exoplasmic to cytoplasmic leaflet of cell membranes, a property crucial for many biological processes. Mutations in P4-ATPases are associated with severe inherited and complex human disorders. We determined the expression, localization and ATPase activity of four variants of ATP8A2, the P4-ATPase associated with the neurodevelopmental disorder known as cerebellar ataxia, impaired intellectual development and disequilibrium syndrome 4 (CAMRQ4). Two variants, G447R and A772P, harboring mutations in catalytic domains, expressed at low levels and mislocalized in cells. In contrast, the E459Q variant in a flexible loop displayed wild-type expression levels, Golgi-endosome localization and ATPase activity. The R1147W variant expressed at 50% of wild-type levels but showed normal localization and activity. These results indicate that the G447R and A772P mutations cause CAMRQ4 through protein misfolding. The E459Q mutation is unlikely to be causative, whereas the R1147W may display a milder disease phenotype. Using various programs that predict protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments, suggesting that such analysis is useful in identifying protein misfolding disease-associated variants.

Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP.

Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

Electrogenic reaction step and phospholipid translocation pathway of the mammalian P4-ATPase ATP8A2.

ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.

Author-level data confirm the widening gender gap in publishing rates during COVID-19.

Publications are essential for a successful academic career, and there is evidence that the COVID-19 pandemic has amplified existing gender disparities in the publishing process. We used longitudinal publication data on 431,207 authors in four disciplines - basic medicine, biology, chemistry and clinical medicine - to quantify the differential impact of COVID-19 on the annual publishing rates of men and women. In a difference-in-differences analysis, we estimated that the average gender difference in publication productivity increased from -0.26 in 2019 to -0.35 in 2020; this corresponds to the output of women being 17% lower than the output of men in 2109, and 24% lower in 2020. An age-group comparison showed a widening gender gap for both early-career and mid-career scientists. The increasing gender gap was most pronounced among highly productive authors and in biology and clinical medicine. Our study demonstrates the importance of reinforcing institutional commitments to diversity through policies that support the inclusion and retention of women in research.

Lack of consideration of sex and gender in COVID-19 clinical studies.

Sex and gender differences impact the incidence of SARS-CoV-2 infection and COVID-19 mortality. Furthermore, sex differences influence the frequency and severity of pharmacological side effects. A large number of clinical trials to develop new therapeutic approaches and vaccines for COVID-19 are ongoing. We investigated the inclusion of sex and/or gender in COVID-19 studies on ClinicalTrials.gov, collecting data for the period January 1, 2020 to January 26, 2021. Here, we show that of the 4,420 registered SARS-CoV-2/COVID-19 studies, 935 (21.2%) address sex/gender solely in the context of recruitment, 237 (5.4%) plan sex-matched or representative samples or emphasized sex/gender reporting, and only 178 (4%) explicitly report a plan to include sex/gender as an analytical variable. Just eight (17.8%) of the 45 COVID-19 related clinical trials published in scientific journals until December 15, 2020 report sex-disaggregated results or subgroup analyses.

Weak evidence of country- and institution-related status bias in the peer review of abstracts.

Research suggests that scientists based at prestigious institutions receive more credit for their work than scientists based at less prestigious institutions, as do scientists working in certain countries. We examined the extent to which country- and institution-related status signals drive such differences in scientific recognition. In a preregistered survey experiment, we asked 4,147 scientists from six disciplines (astronomy, cardiology, materials science, political science, psychology and public health) to rate abstracts that varied on two factors: (i) author country (high status vs lower status in science); (ii) author institution (high status vs lower status university). We found only weak evidence of country- or institution-related status bias, and mixed regression models with discipline as random-effect parameter indicated that any plausible bias not detected by our study must be small in size.

Global citation inequality is on the rise.

Citations are important building blocks for status and success in science. We used a linked dataset of more than 4 million authors and 26 million scientific papers to quantify trends in cumulative citation inequality and concentration at the author level. Our analysis, which spans 15 y and 118 scientific disciplines, suggests that a small stratum of elite scientists accrues increasing citation shares and that citation inequality is on the rise across the natural sciences, medical sciences, and agricultural sciences. The rise in citation concentration has coincided with a general inclination toward more collaboration. While increasing collaboration and full-count publication rates go hand in hand for the top 1% most cited, ordinary scientists are engaging in more and larger collaborations over time, but publishing slightly less. Moreover, fractionalized publication rates are generally on the decline, but the top 1% most cited have seen larger increases in coauthored papers and smaller relative decreases in fractional-count publication rates than scientists in the lower percentiles of the citation distribution. Taken together, these trends have enabled the top 1% to extend its share of fractional- and full-count publications and citations. Further analysis shows that top-cited scientists increasingly reside in high-ranking universities in western Europe and Australasia, while the United States has seen a slight decline in elite concentration. Our findings align with recent evidence suggesting intensified international competition and widening author-level disparities in science.

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

COVID-19 medical papers have fewer women first authors than expected.

The COVID-19 pandemic has resulted in school closures and distancing requirements that have disrupted both work and family life for many. Concerns exist that these disruptions caused by the pandemic may not have influenced men and women researchers equally. Many medical journals have published papers on the pandemic, which were generated by researchers facing the challenges of these disruptions. Here we report the results of an analysis that compared the gender distribution of authors on 1893 medical papers related to the pandemic with that on papers published in the same journals in 2019, for papers with first authors and last authors from the United States. Using mixed-effects regression models, we estimated that the proportion of COVID-19 papers with a woman first author was 19% lower than that for papers published in the same journals in 2019, while our comparisons for last authors and overall proportion of women authors per paper were inconclusive. A closer examination suggested that women's representation as first authors of COVID-19 research was particularly low for papers published in March and April 2020. Our findings are consistent with the idea that the research productivity of women, especially early-career women, has been affected more than the research productivity of men.

Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase.

Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane - in fact, mutations Q925A and Q925G increased the apparent Na+ affinity - but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic.

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

Gender variations in citation distributions in medicine are very small and due to self-citation and journal prestige.

A number of studies suggest that scientific papers with women in leading-author positions attract fewer citations than those with men in leading-author positions. We report the results of a matched case-control study of 1,269,542 papers in selected areas of medicine published between 2008 and 2014. We find that papers with female authors are, on average, cited between 6.5 and 12.6% less than papers with male authors. However, the standardized mean differences are very small, and the percentage overlaps between the distributions for male and female authors are extensive. Adjusting for self-citations, number of authors, international collaboration and journal prestige, we find near-identical per-paper citation impact for women and men in first and last author positions, with self-citations and journal prestige accounting for most of the small average differences. Our study demonstrates the importance of focusing greater attention on within-group variability and between-group overlap of distributions when interpreting and reporting results of gender-based comparisons of citation impact.

Identification and functional analyses of disease-associated P4-ATPase phospholipid flippase variants in red blood cells.

ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry was first detected in red blood cell (RBC) membranes, but the P4-ATPases responsible have not been directly determined. Using affinity-based MS, we show that ATP11C is the only abundant P4-ATPase phospholipid flippase in human RBCs, whereas ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. We also found that ATP11A and ATP11B are present at low levels. Mutations in the gene encoding ATP11C are responsible for blood and liver disorders, but the disease mechanisms are not known. Using heterologous expression, we show that the T415N substitution in the phosphorylation motif of ATP11C, responsible for congenital hemolytic anemia, reduces ATP11C expression, increases retention in the endoplasmic reticulum, and decreases ATPase activity by 61% relative to WT ATP11C. The I355K substitution in the transmembrane domain associated with cholestasis and anemia in mice was expressed at WT levels and trafficked to the plasma membrane but was devoid of activity. We conclude that the T415N variant causes significant protein misfolding, resulting in low protein expression, cellular mislocalization, and reduced functional activity. In contrast, the I355K variant folds and traffics normally but lacks key contacts required for activity. We propose that the loss in ATP11C phospholipid flippase activity coupled with phospholipid scramblase activity results in the exposure of phosphatidylserine on the surface of RBCs, decreasing RBC survival and resulting in anemia.

Asparagine 905 of the mammalian phospholipid flippase ATP8A2 is essential for lipid substrate-induced activation of ATP8A2 dephosphorylation.

The P-type ATPase protein family includes, in addition to ion pumps such as Ca2+-ATPase and Na+,K+-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.

A Darier disease mutation relieves kinetic constraints imposed by the tail of sarco(endo)plasmic reticulum Ca2+-ATPase 2b.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2+-pump reaction cycle. Here, we report that a Darier disease (DD) mutation of SERCA2b that changes a glutamate to a lysine in the cytoplasmic loop between TM8 and TM9 (E917K) relieves these kinetic constraints. We analyzed the effects of this mutation on the overall reaction and the individual partial reactions of the Ca2+ pump compared with the corresponding mutations of the SERCA2a and SERCA1a isoforms, lacking the SERCA2b tail. In addition to a reduced affinity for Ca2+, caused by the mutation in all three isoforms examined, we observed a unique enhancing effect on the turnover rates of ATPase activity and Ca2+ transport for the SERCA2b E917K mutation. This relief of kinetic constraints contrasted with inhibitory effects observed for the corresponding SERCA2a and SERCA1a (E918K) mutations. These observations indicated that the E917K/E918K mutations affect the rate-limiting conformational change in isoform-specific ways and that the SERCA2b mutation perturbs the interactions of TM11 with other SERCA2b regions. Mutational analysis of an arginine in TM7 that interacts with the glutamate in SERCA1a crystal structures suggested that in wildtype SERCA2b, the corresponding arginine (Arg-835) may be involved in mediating the conformational restriction by TM11. Moreover, the E917K mutation may disturb TM11 through the cytoplasmic loop between TM10 and TM11. In conclusion, our findings have identified structural elements of importance for the kinetic constraints imposed by TM11.

One and a half million medical papers reveal a link between author gender and attention to gender and sex analysis.

Gender and sex analysis is increasingly recognized as a key factor in creating better medical research and health care 1-7 . Using a sample of more than 1.5 million medical research papers, our study examined the potential link between women's participation in medical science and attention to gender-related and sex-related factors in disease-specific research. Adjusting for variations across countries, disease topics and medical research areas, we compared the participation of women authors in studies that do and do not involve gender and sex analysis. Overall, our results show a robust positive correlation between women's authorship and the likelihood of a study including gender and sex analysis. These findings corroborate discussions of how women's participation in medical science links to research outcomes, and show the mutual benefits of promoting both the scientific advancement of women and the integration of gender and sex analysis into medical research.

Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase.

Rescue of defective ATP8B1 trafficking by CFTR correctors as a therapeutic strategy for familial intrahepatic cholestasis.

BACKGROUND & AIMS: ATP8B1 deficiency is an autosomal recessive liver disease characterized by intrahepatic cholestasis. ATP8B1 mutation p.I661T, the most frequent mutation in European patients, results in protein misfolding and impaired targeting to the plasma membrane. Similarly, mutations in cystic fibrosis transmembrane conductance regulator (CFTR), associated with cystic fibrosis, impair protein folding and trafficking. The aim of this study was to investigate whether compounds that rescue CFTR F508del trafficking are capable of improving p.I661T-ATP8B1 plasma membrane expression. METHODS: The effect of CFTR corrector compounds on plasma membrane expression of p.I661T-ATP8B1 was evaluated by cell surface biotinylation and immunofluorescence. ATPase activity was evaluated of a purified analogue protein carrying a mutation at the matching position (p.L622T-ATP8A2). RESULTS: The clinically used compounds, 4-phenylbutyric acid (4-PBA), suberoylanilide hydroxamic acid (SAHA) and N-butyldeoxynojirimycin (NB-DNJ) improved p.I661T-ATP8B1 plasma membrane targeting. Compounds C4, C5, C13 and C17 also significantly increased plasma membrane expression of p.I661T-ATP8B1. SAHA and compound C17 upregulated ATP8B1 transcription. p.I661T-ATP8B1 was partly targeted to the canalicular membrane in polarized cells, which became more evident upon treatment with SAHA and/or C4. p.L622T-ATP8A2 showed phospholipid-induced ATPase activity, suggesting that mutations at a matching position in ATP8B1 do not block functionality. Combination therapy of SAHA and compound C4 resulted in an additional improvement of ATP8B1 cell surface abundance. CONCLUSIONS: This study shows that several CFTR correctors can improve trafficking of p.I661T-ATP8B1 to the plasma membrane in vitro. Hence, these compounds may be suitable to be part of a future therapy for ATP8B1 deficiency and other genetic disorders associated with protein misfolding. LAY SUMMARY: Compounds that improve the cellular machinery dealing with protein homeostasis (proteostasis) and allow for proper folding of proteins with (mild) missense mutations are called proteostasis regulators (Balch, Science 2008). Such compounds are potentially of high therapeutic value for many (liver) diseases. In this manuscript, we investigated whether compounds identified in screens as CFTR folding correctors are actually proteostasis regulators and thus have a broader application in other protein folding diseases. Using these compounds, we could indeed show improved trafficking to the (apical) plasma membrane of a mutated ATP8B1 protein, carrying the p.I661T missense mutation. This is the most frequently identified mutation in this rare cholestatic disorder. Importantly, ATP8B1 shows no similarity to CFTR. These data are important in providing support for the concept that rare, genetic liver diseases can potentially be treated using a generalized strategy.