Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

Hypoxia extends lifespan and neurological function in a mouse model of aging.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.

Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis.

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases.

Multiorgan failure in a fatal case of autoimmune hemolytic anemia.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) results in red blood cell destruction by auto-antibodies directed against surface antigens and is rarely fatal. Here we describe a case of AIHA, refractory to both standard and experimental therapies, complicated by multiorgan failure, and rapidly leading to death. CASE REPORT AND RESULTS: A 65 year-old man who presented with progressive dyspnea and jaundice was found to have hemolytic anemia. Diagnostic work-up revealed a positive direct antiglobulin test and a strong pan-reactive antibody in the plasma reacting to a titer of 1:1024 with strongest reactivity at 37 °C Coombs' phase with reagent anti-IgG. The red cell eluate contained a pan-agglutinin. The patient received multiple lines of treatment including glucocorticoids, intravenous immunoglobulin, rituximab, eculizumab, splenectomy and etoposide. Despite these interventions, he continued to experience brisk hemolysis and remained transfusion dependent. Repeat testing on day 16 demonstrated persistent high titer IgG auto-antibodies, suggesting minimal suppressive effect of therapy. His course was complicated by acute renal and liver failure, venous thrombosis, and worsening coagulopathy, and he ultimately died from multiorgan failure on day 18. CONCLUSION: Severe cases of AIHA can result in multiorgan failure and a fatal outcome. The rapid development of liver failure in this setting has been described in only few case reports to date, and represents an important complication for clinicians to be aware of when treating patients with AIHA.

Retrospective cohort studies of repeat donors reveal donor-dependent variability in the recovery of transfused platelets.

BACKGROUND: The in vivo recovery of transfused platelets is variable and often unpredictable. Although many recipient-dependent factors are well described, donor-dependent variables remain poorly understood. STUDY DESIGN AND METHODS: To explore donor-dependent variables we conducted 2 retrospective studies of platelet transfusion outcomes in repeat donors. One study analyzed multiple autologous, radiolabeled platelet transfusions, and a second study analyzed multiple clinical platelet transfusions from a small cohort of repeat donors. RESULTS: In 36 subjects, multiple within-subject determinations of recovery and survival of radiolabeled autologous platelets revealed a relative consistency in platelet recoveries within donors compared to the range of recoveries among donors. Intraclass correlation coefficients for platelet recovery were 43% to 93%. In 524 ABO-compatible clinical platelet transfusions derived from seven donors, a linear mixed-effects model revealed significant donor-dependent differences in corrected count increments for units stored for 4 or 5 days. CONCLUSIONS: These two studies indicate reproducible donor-dependent differences in transfused platelet recovery, suggesting a possible heritable influence on the quality of transfused platelets.

Marked factor V activity elevation in severe COVID-19 is associated with venous thromboembolism.

Coagulopathy causes morbidity and mortality in patients with coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Yet, the mechanisms are unclear and biomarkers are limited. Early in the pandemic, we observed markedly elevated factor V activity in a patient with COVID-19, which led us to measure factor V, VIII, and X activity in a cohort of 102 consecutive inpatients with COVID-19. Contemporaneous SARS-CoV-2-negative controls (n = 17) and historical pre-pandemic controls (n = 260-478) were also analyzed. This cohort represents severe COVID-19 with high rates of ventilator use (92%), line clots (47%), deep vein thrombosis or pulmonary embolism (DVT/PE) (23%), and mortality (22%). Factor V activity was significantly elevated in COVID-19 (median 150 IU/dL, range 34-248 IU/dL) compared to contemporaneous controls (median 105 IU/dL, range 22-161 IU/dL) (P < .001)-the strongest association with COVID-19 of any parameter studied, including factor VIII, fibrinogen, and D-dimer. Patients with COVID-19 and factor V activity >150 IU/dL exhibited significantly higher rates of DVT/PE (16/49, 33%) compared to those with factor V activity ≤150 IU/dL (7/53, 13%) (P = .03). Within this severe COVID-19 cohort, factor V activity associated with SARS-CoV-2 load in a sex-dependent manner. Subsequent decreases in factor V were linked to progression toward DIC and mortality. Together, these data reveal marked perturbations of factor V activity in severe COVID-19, provide links to SARS-CoV-2 disease biology and clinical outcomes, and nominate a candidate biomarker to investigate for guiding anticoagulation therapy in COVID-19.

A physician-scientist preceptorship in clinical and translational research enhances training and mentorship.

BACKGROUND: Dual degree program MD/PhD candidates typically train extensively in basic science research and in clinical medicine, but often receive little formal experience or mentorship in clinical and translational research. METHODS: To address this educational and curricular gap, the University of Wisconsin Medical Scientist Training Program partnered with the University of Wisconsin Institute for Clinical and Translational Research to create a new physician-scientist preceptorship in clinical and translational research. This six-week apprentice-style learning experience-guided by a physician-scientist faculty mentor-integrates both clinical work and a translational research project, providing early exposure and hands-on experience with clinically oriented research and the integrated career of a physician-scientist. Five years following implementation, we retrospectively surveyed students and faculty members to determine the outcomes of this preceptorship. RESULTS: Over five years, 38 students and 36 faculty members participated in the physician-scientist preceptorship. Based on student self-assessments (n = 29, response rate 76%), the course enhanced competency in conducting translational research and understanding regulation of clinical research among other skills. Mentor assessments (n = 17, response rate 47%) supported the value of the preceptorship in these same areas. Based on work during the preceptorship, half of the students produced a peer-reviewed publication or a meeting abstract. At least eleven peer-reviewed manuscripts were generated. The preceptorship also provided a structure for physician-scientist mentorship in the students' clinical specialty of choice. CONCLUSION: The physician-scientist preceptorship provides a new curricular model to address the gap of clinical research training and provides for mentorship of physician-scientists during medical school. Future work will assess the long-term impact of this course on physician-scientist career trajectories.

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis.

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.

Amine-reactive neutron-encoded labels for highly plexed proteomic quantitation.

We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between (13)C and (15)N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference-relative to its nearest neighbor-so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration.

A Gly-zipper motif mediates homodimerization of the transmembrane domain of the mitochondrial kinase ADCK3.

Interactions between α-helices within the hydrophobic environment of lipid bilayers are integral to the folding and function of transmembrane proteins; however, the major forces that mediate these interactions remain debated, and our ability to predict these interactions is still largely untested. We recently demonstrated that the frequent transmembrane association motif GASright, the GxxxG-containing fold of the glycophorin A dimer, is optimal for the formation of extended networks of Cα-H hydrogen bonds, supporting the hypothesis that these bonds are major contributors to association. We also found that optimization of Cα-H hydrogen bonding and interhelical packing is sufficient to computationally predict the structure of known GASright dimers at near atomic level. Here, we demonstrate that this computational method can be used to characterize the structure of a protein not previously known to dimerize, by predicting and validating the transmembrane dimer of ADCK3, a mitochondrial kinase. ADCK3 is involved in the biosynthesis of the redox active lipid, ubiquinone, and human ADCK3 mutations cause a cerebellar ataxia associated with ubiquinone deficiency, but the biochemical functions of ADCK3 remain largely undefined. Our experimental analyses show that the transmembrane helix of ADCK3 oligomerizes, with an interface based on an extended Gly-zipper motif, as predicted by our models. The data provide strong evidence for the hypothesis that optimization of Cα-H hydrogen bonding is an important factor in the association of transmembrane helices. This work also provides a structural foundation for investigating the role of transmembrane association in regulating the biological activity of ADCK3.

Computer vision quantitation of erythrocyte shape abnormalities provides diagnostic, prognostic, and mechanistic insight.

Examination of red blood cell (RBC) morphology in peripheral blood smears can help diagnose hematologic diseases, even in resource-limited settings, but this analysis remains subjective and semiquantitative with low throughput. Prior attempts to develop automated tools have been hampered by their poor reproducibility and limited clinical validation. Here, we present a novel, open-source machine-learning approach (denoted as RBC-diff) to quantify abnormal RBCs in peripheral smear images and generate an RBC morphology differential. RBC-diff cell counts showed high accuracy for single-cell classification (mean AUC, 0.93) and quantitation across smears (mean R2, 0.76 compared with experts, interexperts R2, 0.75). RBC-diff counts were concordant with the clinical morphology grading for 300 000+ images and recovered the expected pathophysiologic signals in diverse clinical cohorts. Criteria using RBC-diff counts distinguished thrombotic thrombocytopenic purpura and hemolytic uremic syndrome from other thrombotic microangiopathies, providing greater specificity than clinical morphology grading (72% vs 41%; P < .001) while maintaining high sensitivity (94% to 100%). Elevated RBC-diff schistocyte counts were associated with increased 6-month all-cause mortality in a cohort of 58 950 inpatients (9.5% mortality for schist. >1%, vs 4.7% for schist; <0.5%; P < .001) after controlling for comorbidities, demographics, clinical morphology grading, and blood count indices. RBC-diff also enabled the estimation of single-cell volume-morphology distributions, providing insight into the influence of morphology on routine blood count measures. Our codebase and expert-annotated images are included here to spur further advancement. These results illustrate that computer vision can enable rapid and accurate quantitation of RBC morphology, which may provide value in both clinical and research contexts.

Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage.

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

Chemotherapy-sparing treatment of haemophagocytic lymphohistiocytosis with intravenous immunoglobulins and corticosteroids.

Haemophagocytic lymphohistiocytosis (HLH) can be a rapidly fatal disease. Current treatment in adults is extrapolated from the HLH-2004 protocol that specifies a regimen of etoposide, dexamethasone and cyclosporine. However, HLH presents as a spectrum of disease severity. A therapeutic challenge arises for milder cases where the harms of potent chemotherapy such as etoposide may outweigh its benefit. We present a case of an adult with HLH who developed significant pancytopenia but was otherwise not critically ill and who responded to treatment with a chemotherapy-sparing approach consisting of intravenous immunoglobulins and corticosteroids alone. The case illustrates that tailored therapy may allow effective treatment of the disorder while minimising therapy-related toxicities.

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.