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Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.

Amyloid fibril formation plays a central role in the pathogenesis of a number of neurodegenerative diseases, including Alzheimer and Parkinson diseases. Transient prefibrillar oligomers forming during the aggregation process, exhibiting a small size and a large hydrophobic surface, can aberrantly interact with a number of molecular targets on neurons, including the lipid bilayer of plasma membranes, resulting in a fatal outcome for the cells. By contrast, the mature fibrils, despite presenting generally a high hydrophobic surface, are endowed with a low diffusion rate and poorly penetrate the interior of the lipid bilayer. However, increasing evidence shows that both intracellular α-synuclein fibrils, as well and as extracellular amyloid-β and β2-microglobulin fibrils, can release oligomers over time that quickly diffuse to reach the membrane of the neighboring cells. The persistent leakage of harmful oligomers from fibrils triggers an ongoing cascade of events resulting in a sustained injury to neurons and glia and also provides aggregates with the ability to cross biological membranes and diffuse between cells or cellular compartments.

The involvement of transactivation response (TAR) DNA-binding protein 43 (TDP-43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. After 12 yr it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full-length protein, which has limited so far the characterization of its structure, function, molecular interactors, and pathobiology. Using a novel protocol we have achieved the purification of the full-length TDP-43, with both a short pectate lyase B tag and a glutathione S-transferase tag, which consisted in its expression in bacteria, solubilization from inclusion bodies, purification under denaturing conditions, refolding, and a final size exclusion chromatography (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and its stability. The protein is pure, as determined with electrophoresis, Western blotting, and mass spectrometry; properly refolded, as revealed by circular dichroism and fluorescence spectroscopies; functional, because it binds to DNA and protein partners; and stable to degradation and aggregation in a physiologic solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer.-Vivoli Vega, M., Nigro, A., Luti, S., Capitini, C., Fani, G., Gonnelli, L., Boscaro, F., Chiti, F. Isolation and characterization of soluble human full-length TDP-43 associated with neurodegeneration.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Studies in vitro show that globular proteins can experience the formation of native-like conformational states able to self-assemble with no need of transitions across the energy barrier for unfolding, and that such processes can lead eventually to the formation of amyloid-like species. Circumstantial evidence collected in vivo suggests that aggregation of native-like states can be a concrete possibility for living organisms and thus more relevant than previously thought. In this review we summarize the key observations collected on the “native-like aggregation” of the acylphosphatase from Sulfolobus solfataricus, a protein that has allowed the direct monitoring and analysis of native-like aggregates for its propensity to form rapidly native-like aggregates and their slow conversion into amyloid-like aggregates.

The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.

Among the many parameters that have been proposed to promote amyloid fibril formation is the pi-stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non-aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation-promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic beta-sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by H-1-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 angstrom resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same beta alpha beta alpha beta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, T-m, of 100.8 degrees C and an unfolding free energy, Delta G(U-F)(H2O), at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Analysis of the Drosophila melanogaster EST data-base led to the characterization of a novel acylphosphatase(AcPDro2). This is coded by the CG18505 (Acyp2) gene andisclearlydistinctfromapreviouslydescribedAcPDrocodedbytheCG16870(Acyp)genefromD. melanogaster.Thetwopro-teins show a 60% homology with both vertebrate isoenzymes.All the residues involved in the catalytic mechanism are con-served. AcPDro2 is a stable enzyme with a correct globularfolded structure. Its activity on benzoylphosphate shows higherK cat but lower K m with respect to AcPDro. It is possible thatAcPDroandAcPDro2genesarenotthedirectancestorofMTand CT vertebrate isoenzymes.- 2003 Federation of European Biochemical Societies. Pub-lished by Elsevier Science B.V. All rights reserved.Keywords: Acylphosphatase; Activity; Protein stability;Ancestor; Drosophila1. IntroductionAcylphosphatase (E.C. 3.6.1.7) is a small cytosolic enzymewhichcatalyzesthehydrolysisofcarboxylphosphatebondofsynthetic and physiological molecules [1]. The role and thefunction of this enzyme in vivo is not yet completely clearthough a number of in vitro evidences show that acylphos-phatasehydrolyticactivityhasaneiectonmembranepumpsthatpresentphosphointermediatesintheircatalyticcycle,sug-gesting a possible role in controlling the ion transport acrossbiologicalmembranes[2^4].Otherevidencesshowanincreas-inglevelofexpressionduringdiierentiationofseveralhumancell lines [5^7]. In addition, it has been demonstrated thatoverexpression of acylphosphatase in HeLa cells induces ap-optosis [8].Acylphosphatase is widely distributed in many tissues ofvertebrate species and it is found as two isoforms, namedmuscle-type (MT) and common-type (CT) sharing a 56% ofsequenceidentity.Thecharacterizationofisoformsfromphy-logenetically representative organisms shows a stable conser-vation of amino acid sequence from remote vertebrates tomammalians [1].Thethree-dimensional structure ofboth isoenzymes, deter-mined by NMR and X-ray crystallography, shows a similarconformation,atypicalK/L globularfoldfoundinotherphos-phate binding proteins [9,10]. Recently, it was demonstratedthat acylphosphatases are able to form protein aggregatesmorphologically similar to those involved in degenerativepathologies such as Alzheimer’s disease and Parkinson’s dis-ease [11]. At present acylphosphatases are considered a goodmodel system for protein aggregation studies [12,13].Site directed mutagenesis demonstrated that the Arg-23conserved residue is responsible for the phosphate bindingand thatthe Asn-41 residue is responsible for catalytic activ-ity. The proposed catalytic mechanism considers the orienta-tion of a catalytic water molecule by the Asn-41 residue to-wards the substrate and the subsequent disruption ofphosphate bond from acyl group and phosphate group [1].Although acylphosphatases have been isolated and charac-terizedonlyfromvertebratespecies,thedetectionoftheacyl-phosphatase activity has been reported in lower organismssuchasbacteriaandinsects.AlsocharacterizedwasaDroso-phila melanogaster acylphosphatase (named AcPDro), as theproduct of a single exon in the Acyp gene located in 2Lchromosome. This enzyme shows a 40% identity and a 66%homologywithbothhumanisoenzymes,thoughpresentinganadditional tail of 22 amino acids at the C-terminus. Since itwas the most phylogenetically distant acylphosphatase thatwas characterized, it was considered as the possible commonancestor for MT and CT vertebrate isoenzymes [14].In this paper we report the characterization of the proteincodedbytheCG18505(Acyp2)gene.ThisworkdemonstratesthattheproductofAcyp2geneisafullyactiveenzyme.Thisisanovelacylphosphatase(AcPDro2),withkineticpropertiesclearlydistinctfromthoseofthepreviouslydescribedAcPDro[14]. The presence of more than one protein with acylphos-phatase activity in D. melanogaster demonstrates that also inthis species more than one form of this enzyme is present.2. Materials and methods

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.

It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases. A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation. Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated. We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP. By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost. These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo.

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.

RT-PCR experiments on RNA from K562 and HepG2 cells and from human placenta led to the isolation of a novel cDNA, a further alternative splicing product of the primary transcript of low M r phosphotyrosine phosphatase (LMW-PTP), already known to produce isoforms 1 and 2. This new transcript represents 15–20% of the total LMW-PTP mRNA in the cell. This novel cDNA codifies for a protein that we have named SV3 (splicing variant 3): the deduced protein sequence presents the first 49 residues identical to those of isoform 1, followed by 24 unrelated amino acids, due to a frameshift introduced at the novel exon-exon boundary. The SV3 protein, expressed in E. coli is enzymatically inactive, most probably because unfolded, as suggested by far-UV circular dichroism (CD) experiments. SV3 protein appears to possess the characteristics of an unstructured polypeptide chain lacking the packing of side chain residues and the secondary structure level that are typical of globular proteins. This protein could represent an inactive variant of the human LMW-PTP.

As structural flexibility is known to be required for enzyme catalysis and pattern recognition and a significant fraction of eukaryotic proteins appear to be unfolded or contain unstructured regions, biological activity of conformational states distinct from fully folded structures could be more common than previously thought. By applying a procedure that allows the recovery of enzymatic activity to be monitored in real time, we show that a non-native state populated transiently during folding of the acylphosphatase from Sulfolobus solfataricus is enzymatically active. The structural characterization of this partially folded state reveals that enzymatic activity is possible even if the catalytic site is structurally heterogeneous, whereas the remainder of the structure acts as a scaffold. These results extend the spectrum of biological functions carried out in the absence of a folded state to include enzyme catalysis.