Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Tumor suppressor PNRC1 blocks rRNA maturation by recruiting the decapping complex to the nucleolus.

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

PA28γ-20S proteasome is a proteolytic complex committed to degrade unfolded proteins.

PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.

SMfinder: Small Molecules Finder for Metabolomics and Lipidomics Analysis.

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis.

eEF2K/eEF2 Pathway Controls the Excitation/Inhibition Balance and Susceptibility to Epileptic Seizures.

Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.

DDK dependent regulation of TOP2A at centromeres revealed by a chemical genetics approach.

In eukaryotic cells the CDC7/DBF4 kinase, also known as DBF4-dependent kinase (DDK), is required for the firing of DNA replication origins. CDC7 is also involved in replication stress responses and its depletion sensitises cells to drugs that affect fork progression, including Topoisomerase 2 poisons. Although CDC7 is an important regulator of cell division, relatively few substrates and bona-fide CDC7 phosphorylation sites have been identified to date in human cells. In this study, we have generated an active recombinant CDC7/DBF4 kinase that can utilize bulky ATP analogues. By performing in vitro kinase assays using benzyl-thio-ATP, we have identified TOP2A as a primary CDC7 substrate in nuclear extracts, and serine 1213 and serine 1525 as in vitro phosphorylation sites. We show that CDC7/DBF4 and TOP2A interact in cells, that this interaction mainly occurs early in S-phase, and that it is compromised after treatment with CDC7 inhibitors. We further provide evidence that human DBF4 localises at centromeres, to which TOP2A is progressively recruited during S-phase. Importantly, we found that CDC7/DBF4 down-regulation, as well S1213A/S1525A TOP2A mutations can advance the timing of centromeric TOP2A recruitment in S-phase. Our results indicate that TOP2A is a novel DDK target and have important implications for centromere biology.

SEPN1, an endoplasmic reticulum-localized selenoprotein linked to skeletal muscle pathology, counteracts hyperoxidation by means of redox-regulating SERCA2 pump activity.

Selenoprotein N (SEPN1) is a broadly expressed resident protein of the endoplasmic reticulum (ER) whose loss-of-function inexplicably leads to human muscle disease. We found that SEPN1 levels parallel those of endoplamic reticulum oxidoreductin 1 (ERO1), an ER protein thiol oxidase, and that SEPN1's redox activity defends the ER from ERO1-generated peroxides. Moreover, we have defined the redox-regulated interactome of SEPN1 and identified the ER calcium import SERCA2 pump as a redox-partner of SEPN1. SEPN1 enhances SERCA2 activity by reducing luminal cysteines that are hyperoxidized by ERO1-generated peroxides. Cells lacking SEPN1 are hypersensitive to ERO1 overexpression and conspicuously defective in ER calcium re-uptake. After being muscle-transduced with an adeno-associated virus driving ERO1α, SEPN1 knockout mice unmasks a myopathy that resembles the dense core disease due to human mutations in SEPN1, whereas the combined attenuation of ERO1α and SEPN1 enhances cell fitness. These observations reveal the involvement of SEPN1 in ER redox and calcium homeostasis and that an ERO1 inhibitor, restoring redox-dependent calcium homeostasis, may ameliorate the myopathy of SEPN1 deficiency.

Predictive value of a proteomic signature in patients with non-small-cell lung cancer treated with second-line erlotinib or chemotherapy (PROSE): a biomarker-stratified, randomised phase 3 trial.

BACKGROUND: An established multivariate serum protein test can be used to classify patients according to whether they are likely to have a good or poor outcome after treatment with EGFR tyrosine-kinase inhibitors. We assessed the predictive power of this test in the comparison of erlotinib and chemotherapy in patients with non-small-cell lung cancer. METHODS: From Feb 26, 2008, to April 11, 2012, patients (aged ≥18 years) with histologically or cytologically confirmed, second-line, stage IIIB or IV non-small-cell lung cancer were enrolled in 14 centres in Italy. Patients were stratified according to a minimisation algorithm by Eastern Cooperative Oncology Group performance status, smoking history, centre, and masked pretreatment serum protein test classification, and randomly assigned centrally in a 1:1 ratio to receive erlotinib (150 mg/day, orally) or chemotherapy (pemetrexed 500 mg/m(2), intravenously, every 21 days, or docetaxel 75 mg/m(2), intravenously, every 21 days). The proteomic test classification was masked for patients and investigators who gave treatments, and treatment allocation was masked for investigators who generated the proteomic classification. The primary endpoint was overall survival and the primary hypothesis was the existence of a significant interaction between the serum protein test classification and treatment. Analyses were done on the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT00989690. FINDINGS: 142 patients were randomly assigned to chemotherapy and 143 to erlotinib, and 129 (91%) and 134 (94%), respectively, were included in the per-protocol analysis. 88 (68%) patients in the chemotherapy group and 96 (72%) in the erlotinib group had a proteomic test classification of good. Median overall survival was 9·0 months (95% CI 6·8-10·9) in the chemotherapy group and 7·7 months (5·9-10·4) in the erlotinib group. We noted a significant interaction between treatment and proteomic classification (pinteraction=0·017 when adjusted for stratification factors; pinteraction=0·031 when unadjusted for stratification factors). Patients with a proteomic test classification of poor had worse survival on erlotinib than on chemotherapy (hazard ratio 1·72 [95% CI 1·08-2·74], p=0·022). There was no significant difference in overall survival between treatments for patients with a proteomic test classification of good (adjusted HR 1·06 [0·77-1·46], p=0·714). In the group of patients who received chemotherapy, the most common grade 3 or 4 toxic effect was neutropenia (19 [15%] vs one [<1%] in the erlotinib group), whereas skin toxicity (one [<1%] vs 22 [16%]) was the most frequent in the erlotinib group. INTERPRETATION: Our findings indicate that serum protein test status is predictive of differential benefit in overall survival for erlotinib versus chemotherapy in the second-line setting. Patients classified as likely to have a poor outcome have better outcomes on chemotherapy than on erlotinib. FUNDING: Italian Ministry of Health, Italian Association of Cancer Research, and Biodesix.

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.

SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA.

Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca2+ levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.

Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.

Urinary secretion and extracellular aggregation of mutant uromodulin isoforms.

Uromodulin is exclusively expressed in the thick ascending limb and is the most abundant protein secreted in urine where it is found in high-molecular-weight polymers. Its biological functions are still elusive, but it is thought to play a protective role against urinary tract infection, calcium oxalate crystal formation, and regulation of water and salt balance in the thick ascending limb. Mutations in uromodulin are responsible for autosomal-dominant kidney diseases characterized by defective urine concentrating ability, hyperuricemia, gout, tubulointerstitial fibrosis, renal cysts, and chronic kidney disease. Previous in vitro studies found retention in the endoplasmic reticulum as a common feature of all uromodulin mutant isoforms. Both in vitro and in vivo we found that mutant isoforms partially escaped retention in the endoplasmic reticulum and reached the plasma membrane where they formed large extracellular aggregates that have a dominant-negative effect on coexpressed wild-type protein. Notably, mutant uromodulin excretion was detected in patients carrying uromodulin mutations. Thus, our results suggest that mutant uromodulin exerts a gain-of-function effect that can be exerted by both intra- and extracellular forms of the protein.

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF121, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF121, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF121 and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF121. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.

Mutant ferritin L-chains that cause neurodegeneration act in a dominant-negative manner to reduce ferritin iron incorporation.

Nucleotide insertions that modify the C terminus of ferritin light chain (FTL) cause neurodegenerative movement disorders named neuroferritinopathies, which are inherited with dominant transmission. The disorders are characterized by abnormal brain iron accumulation. Here we describe the biochemical and crystallographic characterization of pathogenic FTL mutant p.Phe167SerfsX26 showing that it is a functional ferritin with an altered conformation of the C terminus. Moreover we analyze functional and stability properties of ferritin heteropolymers made of 20-23 H-chains and 1-4 L-chains with representative pathogenic mutations or the last 10-28 residues truncated. All the heteropolymers containing the pathogenic or truncated mutants had a strongly reduced capacity to incorporate iron, both when expressed in Escherichia coli, and in vitro when iron was supplied as Fe(III) in the presence of ascorbate. The mutations also reduced the physical stability of the heteropolymers. The data indicate that even a few mutated L-chains are sufficient to alter the permeability of 1-2 of the 6 hydrophobic channels and modify ferritin capacity to incorporate iron. The dominant-negative action of the mutations explains the dominant transmission of the disorder. The data support the hypothesis that hereditary ferritinopathies are due to alterations of ferritin functionality and provide new input on the mechanism of the function of isoferritins.

Critical role of flanking residues in NGR-to-isoDGR transition and CD13/integrin receptor switching.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of alphavbeta3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alphavbeta3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alphavbeta3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alphavbeta5, alphavbeta6, alphavbeta8, and alpha5beta1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.

Catalytic properties of the retinal rod outer segment disk ADP-ribosyl cyclase.

Cyclic ADP-ribose (cADPR) is a second messenger modulating intracellular calcium levels. We have previously described a cADPR-dependent calcium signaling pathway in bovine rod outer segments (ROS), where calcium ions play a pivotal role. ROS ADP-ribosyl cyclase (ADPR-cyclase) was localized in the membrane fraction. In the present work, we examined the properties of the disk ADPR-cyclase through the production of cyclic GDP-ribose from the NAD(+) analogue NGD(+). The enzyme displayed an estimated K(m) for NGD(+) of 12.5 ± 0.3 µM, a V(max) of 26.50 ± 0.70 pmol cyclic GDP-ribose synthesized/min/mg, and optimal pH of 6.5. The effect of divalent cations (Zn(2+), Cu(2+), and Ca(2+)) was also tested. Micromolar Zn(2+) and Cu(2+) inhibited the disk ADPR-cyclase activity (half maximal inhibitory concentration, IC50=1.1 and 3.6 µM, respectively). By contrast, Ca(2+) ions had no effect. Interestingly, the properties of the intracellular membrane-associated ROS disk ADPR-cyclase are more similar to those of the ADPR-cyclase found in CD38-deficient mouse brain, than to those of CD38 or CD157. The novel intracellular mammalian ADPR-cyclase would elicit Ca(2+) release from the disks at various rates in response to change in free Ca(2+) concentrations, caused by light versus dark adaptation, in fact there was no difference in disk ADPR-cyclase activity in light or dark conditions. Data suggest that disk ADPR-cyclase may be a potential target of retinal toxicity of Zn(2+) and may shed light to the role of Cu(2+) and Zn(2+) deficiency in retina.

Opti-nQL: An Optimized, Versatile and Sensitive Nano-LC Method for MS-Based Lipidomics Analysis.

Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Human iPSC-based neurodevelopmental models of globoid cell leukodystrophy uncover patient- and cell type-specific disease phenotypes.

Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of ß-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)-derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD.

Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis.

Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo.