Structure and function of Plasmodium actin II in the parasite mosquito stages.

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.

Separase prevents genomic instability by controlling replication fork speed.

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis.

Src inhibitors modulate frataxin protein levels.

Defective expression of frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect frataxin expression. We found that frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of frataxin but are ineffective on a non-phosphorylatable frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase frataxin expression and rescue the aconitase defect in frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote frataxin accumulation, suggesting their possible use as therapeutics in FRDA.

Gaucher disease due to saposin C deficiency is an inherited lysosomal disease caused by rapidly degraded mutant proteins.

Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation. Five different mutations (i.e. p.C315S, p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F) have been identified to underlie Sap C deficiency. The molecular mechanism by which these mutations affect Sap C function, however, has not been delineated in detail. Here, we characterized biochemically and functionally four of these gene lesions. We show that all Sap C mutants are efficiently produced, and exhibit lipid-binding properties, modulatory behavior on GCase activity and subcellular localization comparable with those of the wild-type protein. We then delineated the structural rearrangement of these mutants, documenting that most proteins assume diverse aberrant disulfide bridge arrangements, which result in a substantial diminished half-life, and rapid degradation via autophagy. These findings further document the paramount importance of disulfide bridges in the stability of Sap C and provide evidence that accelerated degradation of the Sap C mutants is the underlying pathogenetic mechanism of Sap C deficiency.

Native metastable prefibrillar oligomers are the most neurotoxic species among amyloid aggregates.

Many proteins belonging to the amyloid family share the tendency to misfold and aggregate following common steps, and display similar neurotoxicity. In the aggregation pathway different kinds of species are formed, including several types of oligomers and eventually mature fibers. It is now suggested that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. Many kinds of aggregates have been described to exist in a metastable state and in equilibrium with monomers. Up to now it is not clear whether a specific structure is at the basis of the neurotoxicity. Here we characterized, starting from the early aggregation stages, the oligomer populations formed by an amyloid protein, salmon calcitonin (sCT), chosen due to its very slow aggregation rate. To prepare different oligomer populations and characterize them by means of photoinduced cross-linking SDS-PAGE, Energy Filtered-Transmission Electron Microscopy (EF-TEM) and Circular Dichroism (CD) spectroscopy, we used Size Exclusion Chromatography (SEC), a technique that does not influence the aggregation process leaving the protein in the native state. Taking advantage of sCT low aggregation rate, we characterized the neurotoxic potential of the SEC-separated, non-crosslinked fractions in cultured primary hippocampal neurons, analyzing intracellular Ca(2+) influx and apoptotic trend. We provide evidence that native, globular, metastable, prefibrillar oligomers (dimers, trimers and tetramers) were the toxic species and that low concentrations of these aggregates in the population was sufficient to render the sample neurotoxic. Monomers and other kind of aggregates, such as annular or linear protofibers and mature fibers, were totally biologically inactive.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse.

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes.

T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal cell death by apoptosis and impairment of autophagy pathway. In the present study we report the presence of specific antibodies to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. We also found a significant association between the presence of these autoantibodies and hematologic manifestations occurring in these patients. Investigating the possible implication of anti-D4GDI autoantibodies in SLE pathogenesis or progression, we found that these antibodies were capable of binding D4GDI expressed at the lymphocyte surface and triggering a series of subcellular events, including Rho GTPase activation. These antibodies were also able to induce autophagy in T cells from both healthy donors and SLE patients, but only those negative to these antibodies. We can conclude that anti-D4GDI autoantibodies could be capable of triggering important responses in T cells such as cytoskeleton remodeling and autophagy pathway and that, in SLE patients, the chronic exposure to these specific autoantibodies could lead to the selection of autophagy-resistant T cell clones contributing to the pathogenesis of the disease.

HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation.

HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354.

Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles.

Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr(273) in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr(354) as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: relevance to MLC disease pathogenesis.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

Phosphorylation of SRSF1 is modulated by replicational stress.

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.

Embryonic and larval exposure to propylparaben induces developmental and long-term neurotoxicity in zebrafish model.

Parabens are preservatives found in cosmetics, processed foods, and medications. The harmful repercussions on the central nervous system by one of the most common parabens, propylparaben (PrP), are yet unknown, especially during development. In this study, the neurodevelopmental effects of PrP and long-term neurotoxicity were investigated in the zebrafish model, using an integrated approach. Zebrafish embryos were exposed to two different concentrations of PrP (10 and 1000 µg/L), then larvae were examined for their behavioral phenotypes (open-field behavior, startle response, and circadian rhythmicity) and relevant brain markers (cyp19a1b, pax6a, shank3a, and gad1b). Long-term behavioral and cognitive impacts on sociability, cerebral functional asymmetry and thigmotaxis were also examined on juveniles at 30 dpf and 60 dpf. Moreover, proteomics and gene expression analysis were assessed in brains of 60 dpf zebrafish. Interestingly, thigmotaxis was decreased by the high dose in larvae and increased by the low dose in juveniles. The expression of shank3a and gad1b genes was repressed by both PrP concentrations pointing to possible effects of PrP on neurodevelopment and synaptogenesis. Proteomics analysis evidenced alterations related to brain development and lipid metabolism. Overall, the results demonstrated that early-life exposure to PrP promotes developmental and persistent neurobehavioral alterations in the zebrafish model, affecting genes and protein levels possibly associated with brain diseases.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

Giardia duodenalis 14-3-3 protein is polyglycylated by a tubulin tyrosine ligase-like member and deglycylated by two metallocarboxypeptidases.

The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis.

An insight into the abundant proteome of 46BR.1G1 fibroblasts deficient of DNA ligase I.

This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.

The Nitrobenzoxadiazole Derivative NBDHEX Behaves as Plasmodium falciparum Gametocyte Selective Inhibitor with Malaria Parasite Transmission Blocking Activity.

This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C.

Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.

Structural basis of ubiquitination mediated by protein splicing in early Eukarya.

BACKGROUND: Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5). METHODS: We solved the X-ray structures of BIL2 in both the inactive and unprecedented, zinc-induced active, forms. Then, we characterized by mass spectrometry the BUBL splicing products in the absence and in the presence of T.thRas-GTPase. Finally, we investigated the effect of ubiquitination on T.thRas-GTPase by molecular dynamics simulations. RESULTS: The structural analysis demonstrated that zinc-induced conformational change activates protein splicing. Moreover, mass spectrometry characterization of the splicing products shed light on the possible function of BIL2, which operates as a "single-ubiquitin-dispensing-platform", allowing the conjugation, via isopeptide bond formation (K(εNH2)-C-ter), of ubl4 to either ubl5 or T.thRas-GTPase. Lastly, we demonstrated that T.thRas-GTPase ubiquitination occurs in proximity of the nucleotide binding pocket and stabilizes the protein active state. CONCLUSIONS: We demonstrated that BIL2 is activated by zinc and that protein splicing induced by this intein does not take place through classical or aminolysis mechanisms but via formation of a covalent isopeptide bond, causing the ubiquitination of endogenous substrates such as T.thRas-GTPase. GENERAL SIGNIFICANCE: In this "enzyme-free" ubiquitination mechanism the isopeptide formation, which canonically requires E1-E2-E3 enzymatic cascade and constitutes the alphabet of ubiquitin biology, is achieved in a single, concerted step without energy consumption.