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Although 99mTc is not an ideal Auger electron (AE) emitter for Targeted Radionuclide Therapy (TRT) due to its relatively low Auger electron yield, it can be considered a readily available “model” radionuclide useful to validate the design of new classes of AE-emitting radioconjugates. With this in mind, we performed a detailed study of the radiobiological effects and mechanisms of cell death induced by the dual-targeted radioconjugates 99mTc-TPP-BBN and 99mTc-AO-BBN (TPP = triphenylphosphonium; AO = acridine orange; BBN = bombesin derivative) in human prostate cancer PC3 cells. 99mTc-TPP-BBN and 99mTc-AO-BBN caused a remarkably high reduction of the survival of PC3 cells when compared with the single-targeted congener 99mTc-BBN, leading to an augmented formation of γH2AX foci and micronuclei. 99mTc-TPP-BBN also caused a reduction of the mtDNA copy number, although it enhanced the ATP production by PC3 cells. These differences can be attributed to the augmented uptake of 99mTc-TPP-BBN in the mitochondria and enhanced uptake of 99mTc-AO-BBN in the nucleus, allowing the irradiation of these radiosensitive organelles with the short path-length AEs emitted by 99mTc. In particular, the results obtained for 99mTc-TPP-BBN reinforce the relevance of targeting the mitochondria to promote stronger radiobiological effects by AE-emitting radioconjugates.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein–protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

Mutations in the CFTR anion channel cause cystic fibrosis (CF) and have also been related to higher cancer incidence. Previously we proposed that this is linked to an emerging role of functional CFTR in protecting against epithelial–mesenchymal transition (EMT). However, the pathways bridging dysfunctional CFTR to EMT remain elusive. Here, we applied systems biology to address this question. Our data show that YAP1 is aberrantly active in the presence of mutant CFTR, interacting with F508del, but not with wt-CFTR, and that YAP1 knockdown rescues F508del-CFTR processing and function. Subsequent analysis of YAP1 interactors and roles in cells expressing either wt- or F508del-CFTR reveal that YAP1 is an important mediator of the fibrotic/EMT processes in CF. Alongside, five main pathways emerge here as key in linking mutant CFTR to EMT, namely, (1) the Hippo pathway; (2) the Wnt pathway; (3) the TGFβ pathway; (4) the p53 pathway; and (5) MYC signaling. Several potential hub proteins which mediate the crosstalk among these pathways were also identified, appearing as potential therapeutic targets for both CF and cancer.

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region&mdash, the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region&mdash, regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing &Delta, RE and &Delta, RI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that &Delta, RI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

The development of pharmacologically active compounds based on bis(thiosemicarbazones) (BTSC) and on their coordination to metal centers constitutes a promising field of research. We have recently explored this class of ligands and their Cu(II) complexes for the design of cancer theranostics agents with enhanced uptake by tumoral cells. In the present work, we expand our focus to aliphatic and aromatic BTSC Zn(II) complexes bearing piperidine/morpholine pendant arms. The new complexes ZnL1–ZnL4 were characterized by a variety of analytical techniques, which included single-crystal X-ray crystallography for ZnL2 and ZnL3. Taking advantage of the fluorescent properties of the aromatic complexes, we investigated their cellular uptake kinetics and subcellular localization. Furthermore, we tried to elucidate the mechanism of action of the cytotoxic effect observed in human cancer cell line models. The results show that the aliphatic complexes (ZnL1 and ZnL2) have a symmetrical structure, while the aromatic counterparts (ZnL3 and ZnL4) have an asymmetrical nature. The cytotoxic activity was higher for the aromatic BTSC complexes, as well as the cellular uptake, evaluated by measurement of intracellular Zn accumulation. Among the most active complexes, ZnL3 presented the fastest uptake kinetics and lysosomal localization assessed by live-cell microscopy. Detailed studies of its impact on cellular production of reactive oxygen species and impairment of lysosomal membrane integrity reinforced the influence of the pendant piperidine in the biological performance of aromatic BTSC Zn(II) complexes.

BACKGROUND: For most of the >2,000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717–2A>G (c.1585–2A>G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717–2A>G leads to alternative splicing generating

Mutations associated with cystic fibrosis (CF) have complex effects on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common CF mutation, F508del, disrupts the processing to and stability at the plasma membrane and function as a Cl- channel. CFTR is surrounded by a dynamic network of interacting components, referred to as the CFTR Functional Landscape, that impact its synthesis, folding, stability, trafficking and function. CFTR interacting proteins can be manipulated by functional genomic approaches to rescue the trafficking and functional defects characteristic of CF. Here we review recent efforts to elucidate the impact of genetic variation on the ability of the nascent CFTR polypeptide to interact with the proteostatic environment. We also provide an overview of how specific components of this protein network can be modulated to rescue the trafficking and functional defects associated with the F508del variant of CFTR. The identification of novel proteins playing key roles in the processing of CFTR could pave the way for their use as novel therapeutic targets to provide synergistic correction of mutant CFTR for the greater benefit of individuals with CF.

Background and Purpose: Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ABC transporter functioning as a channel. Unique to CFTR are two highly conformationally dynamic regions: the regulatory extension (RE) and regulatory insertion (RI). Removal of the latter rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation.We aimed here to assess the impact of RE removal (alone or with RI or genetic revertants) on F508del-CFTR traffic and how CFTR modulator drugs corrector VX-809/lumacaftor and potentiator VX-770/ivacaftor rescue these combined variants so as to gain insight into the mechanism of action (MoA) of these drugs.Experimental Approach. We generated ∆RE and ∆RI CFTR variants (with and without genetic revertants) by site-directed mutagenesis and used them to stably transfect BHK cell lines. We studied CFTR expression and stability by Western blotting and pulse-chase respectively, plasma membrane levels by cell surface biotinylation and channel activity by the iodide efflux technique.Key Results. Our data demonstrate that ∆RI significantly enhanced rescue of F508del-CFTR by VX-809. Thus, while the presence of the regulatory insertion seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, thus suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. Nevertheless, this negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the regulatory extension which also leads to the stabilization of this mutant. We thus propose that, despite both these regions being conformationally active, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.Supporting Information: Additional figures with supplementary data

Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

A novel role for S100B, a recognized brain distress marker, as a chaperone-like suppressor of Aβ42 aggregation and toxicity., Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer’s disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface–catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

Secretory traffic became a topical field because many important cell regulators are plasma membrane proteins (transporters, channels, receptors), being thus key targets in biomedicine and drug discovery. Cystic fibrosis (CF), caused by defects in a single gene encoding the CF transmembrane conductance regulator (CFTR), constitutes the most common of rare diseases and certainly a paradigmatic one.Here we focus on five different approaches that allow biochemical and cellular characterization of CFTR from its co-translational insertion into the ER membrane to its delivery to the plasma membrane.

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Protein misfolding and conformational changes are a cornerstone of neurodegenerative diseases involving formation and deposition of toxic protein oligomers. Although mutations favor protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a role. Metal ions such as calcium, zinc and copper are key players in brain neurobiology, their homeostasis is altered in most neurodegenerative conditions and they are found within proteinaceous inclusions from patients. In this review we will elucidate the intricate interplay between protein (mis)folding and metal ions, discussing how metals modulate protein folding and influence protein energetics, with specific attention on conformational changes and structural fluctuations. In particular, the influence of metal ion dyshomeostasis during neurodegeneration and the effects of the unique physical and chemical properties at the synaptic environment will be discussed in the context of protein deposition. These interactions will be illustrated by specific examples of proteins involved in neurodegenerative diseases including α-synuclein, tau, superoxide dismutase 1, the prion protein and the amyloid-β peptide. With this approach we aim to systematize the effects of metal ions on protein conformers and illustrate pathways through which they modulate protein aggregation, under different conceptual mechanisms that bridge protein structure, metallochemistry and neurobiology.

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus ( Hn SOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The Hn SOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the Hn SOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens ( Aa SOR). The melting point of the Hn SOR was ≈20°C lower than that of the Aa SOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.

Background New therapies modulating defective CFTR have started to hit the clinic and others are in trial or under development. The endeavour of drug discovery for CFTR protein rescue is however difficult one since over 2000 mutations have been reported. For most of these, especially the rare ones, the associated defects, the respective functional class and their responsiveness to available modulators are still unknown. Our aim here was to characterize the rare R560S mutation using patient-derived materials (rectal biopsies and intestinal organoids) from one CF individual homozygous for this mutation, in parallel with cellular models expressing R560S-CFTR and to assess the functional and biochemical responses to CFTR modulators. Methods Intestinal organoids were prepared from rectal biopsies and analysed by RT-PCR (to assess CFTR mRNA), by Western blot (to assess CFTR protein) and by forskolin-induced swelling (FIS) assay. A novel cell line expressing R560S-CFTR was generated by stably transducing the CFBE parental cell line and used to assess R560S-CFTR processing and function. Both intestinal organoids and the cellular model were used to assess efficacy of CFTR modulators in rescuing this mutation. Results Our results show that: R560S does not affect CFTR mRNA splicing; R560S affects CFTR protein processing, totally abrogating the production of its mature form; R560S-CFTR evidences no function as a Cl− channel; and none of the modulators tested rescued R560S-CFTR processing or function. Conclusion Altogether, these results indicate that R560S is a class II mutation. However, unlike F508del, it cannot be rescued by any of the CFTR modulators tested.

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

The EF-hand protein S100A2 is a cell cycle regulator involved in tumorigenesis, acting through regulation of the p53 activation state. Metal ion-free S100A2 is homodimeric and contains two Ca(2+)-binding sites and two Zn(2+)-binding sites per subunit, whereby the Zn(2+) ion binding to one of the sites is coordinated by residues from two homodimers. The effect of selective binding of these metal ions was investigated using site-specific mutants which lacked one or both zinc sites. CD analysis of secondary structure changes on metallation showed that Zn(2+) binding was associated with a decrease in the secondary structure content, whereas Ca(2+) had the opposite effect in two of the three S100A2 mutants studied. The energy of unfolding (DeltaG(U)) of the apo wild-type S100A2 was determined to be 89.9 kJ mol(-1), and the apparent midpoint transition temperature (T(m)(app))) was 58.4 degrees C. In addition, a detailed study of the urea and thermal unfolding of the S100A2 mutants in different metallation states (apo, Zn(2+) and Ca(2+)) was performed. Thermal denaturation experiments showed that Zn(2+) acts as a destabilizer and Ca(2+) as a stabilizer of the protein conformation. This suggests a synergistic effect between metal binding, protein stability and S100A2 biological activity, according to which Ca(2+) activates and stabilizes the protein, the opposite being observed on Zn(2+) binding.

Secretory traffic became a topical field because many important cell regulators are plasma membrane proteins (transporters, channels, receptors), being thus key targets in biomedicine and drug discovery. Cystic fibrosis (CF), caused by defects in a single gene encoding the CF transmembrane conductance regulator (CFTR), constitutes the most common of rare diseases and certainly a paradigmatic one.Here we focus on five different approaches that allow biochemical and cellular characterization of CFTR from its co-translational insertion into the ER membrane to its delivery to the plasma membrane.

Journal of Proteome Research (2006)5: 2720-2726 A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70-97°C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96h incubation at 90°C), and proceeded with analysis of the remaining proteins by combining one and two dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. A total of 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism’s ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.

Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking – Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.

S100 proteins are small EF-hand Ca2+-binding proteins involved in diverse cellular processes such as cell survival, proliferation and differentiation. Many S100 proteins also bind additional metal ions which modulate their folding and activity. The expression levels of many S100 proteins are significantly increased in cancer, neurodegenerative, inflammatory and autoimmune diseases. Recently, S100A8/A9 were identified as major amyloidogenic proteins in corpora amylacea inclusions of prostate cancer patients and found to undergo metal-mediated amyloid oligomerization and fibrillation in vitro. The amyloidogenic potential has also been found in other S100 family members, suggesting that amyloid-like assemblies may play an important role in S100 biologic activity in health and disease. In this chapter we address the structural diversity of S100 aggregates, as revealed by high-resolution bioimaging techniques. We start by overviewing the structural diversity of individual S100 proteins and their functional oligomers; this is followed by analysis of their amyloidogenic aggregation potential, general characterization of S100 amyloids, and the role of metal ions in aggregation pathways. The morphologic diversity of the aggregates formed by different types of S100 protein is illustrated by electron and atomic force microscopy data. The chapter ends by overviewing the amyloid formation by S100A8/A9 in the aging prostate, showing how microscopy techniques can be used to characterize in vitro and ex vivo amyloids.

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump1 is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump1 is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump1 in E. coli for biophysical and structural analysis.We show that recombinant Ump1 is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump1 shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.

The S100 proteins are a large family of 10-12 kDa EF-hand signaling proteins that bind calcium, and in some cases zinc and copper, functioning as central regulators in a diversity of cellular processes. These proteins have tissue, cell, and subcellular-specific expression patterns, and many have an extracellular function. Altogether, these properties underlie their functional diversity and involvement in several pathological conditions including cancer, inflammation, and neurodegeneration. S100 proteins exhibit considerable structural plasticity, being able to exist as monomers or assemble into dimers, higher oligomers, and amyloids, frequently in a metal-dependent manner. Many of these oligomers are functionally relevant, and S100 amyloids have been recently found in prostatic inclusions. Here, we report experimental procedures for the isolation and quantitation of S100 oligomers from tissues, purification of recombinant human S100 protein for assays and use as standards, and an amyloidogenesis assay that allows monitoring the formation of S100 β-oligomers and amyloids in apo- and metal-bound S100 proteins.

The S100 proteins are a large family of 10-12 kDa EF-hand signaling proteins that bind calcium, and in some cases zinc and copper, functioning as central regulators in a diversity of cellular processes. These proteins have tissue, cell, and subcellular-specific expression patterns, and many have an extracellular function. Altogether, these properties underlie their functional diversity and involvement in several pathological conditions including cancer, inflammation, and neurodegeneration. S100 proteins exhibit considerable structural plasticity, being able to exist as monomers or assemble into dimers, higher oligomers, and amyloids, frequently in a metal-dependent manner. Many of these oligomers are functionally relevant, and S100 amyloids have been recently found in prostatic inclusions. Here, we report experimental procedures for the isolation and quantitation of S100 oligomers from tissues, purification of recombinant human S100 protein for assays and use as standards, and an amyloidogenesis assay that allows monitoring the formation of S100 β-oligomers and amyloids in apo- and metal-bound S100 proteins.

The enhanced stability of a mesophilic metalloprotein was assessed using biophysical spectroscopies. Significant local structural interconversions during thermal insult account for a reorganization of the protein scaffold, without disturbing the active metal site. This cushioning mechanism is proposed to be a generic property of metalloproteins contributing to enhanced stability.

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

Journal of Biological Inorganic Chemistry (2010)15: 271-281 Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe-S proteins contain a highly conserved all-beta fold, which harbors a [2Fe-2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX(2-3)C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe-2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe-2S] Rieske center. The cluster has a redox potential of +48 mV (25ºC and pH 7) and a pKa of 10.1 +/- 0.2. These shift to +77 mV and 8.9 +/- 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (Tm = 99ºC, pH 7.0), but it becomes destabilized upon disulfide reduction (DeltaTm = -9ºC, DeltaCm = -0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.

The EF-hand protein S100A2 is a cell cycle regulator involved in tumorigenesis, acting through regulation of the p53 activation state. Metal ion-free S100A2 is homodimeric and contains two Ca(2+)-binding sites and two Zn(2+)-binding sites per subunit, whereby the Zn(2+) ion binding to one of the sites is coordinated by residues from two homodimers. The effect of selective binding of these metal ions was investigated using site-specific mutants which lacked one or both zinc sites. CD analysis of secondary structure changes on metallation showed that Zn(2+) binding was associated with a decrease in the secondary structure content, whereas Ca(2+) had the opposite effect in two of the three S100A2 mutants studied. The energy of unfolding (DeltaG(U)) of the apo wild-type S100A2 was determined to be 89.9 kJ mol(-1), and the apparent midpoint transition temperature (T(m)(app))) was 58.4 degrees C. In addition, a detailed study of the urea and thermal unfolding of the S100A2 mutants in different metallation states (apo, Zn(2+) and Ca(2+)) was performed. Thermal denaturation experiments showed that Zn(2+) acts as a destabilizer and Ca(2+) as a stabilizer of the protein conformation. This suggests a synergistic effect between metal binding, protein stability and S100A2 biological activity, according to which Ca(2+) activates and stabilizes the protein, the opposite being observed on Zn(2+) binding.

Objectives: To compare Cystic Fibrosis (CF) severity with NOS-1 polymorphisms (pol.) (AAT, TG1 and TG2) and CFTR interaction. Methods: 181 patients included. The NOS-1 pol. was analyzed by Megabace1000® sequencer. Variables: sex, race, scores [Shwachman-Kulczycki, Kanga and Bhalla (EB)], BMI, age, diagnosis age, first symptoms (digestive and pulmonary), first P. aeruginosa, microorganisms (P. aeruginosa mucoid and non-mucoid, A. xylosoxidans, S. aureus, B. cepacia), SaO2, spirometry and comorbidities (nasal polyps, osteoporosis, meconium ileus, pancreatic insufficiency, diabetes mellitus). Statistical analysis: MDR2.0 and MDRPT0.4.7 software. Numerical data: classified by the median. Categorical data: presence or absence (bacteria and comorbidities). Multiple genes analysis in disease with clinical variation, like CF, allows greater understanding of phenotypic diversity. The analysis of all alleles for NOS-1 and CFTR did not show association. The same was observed to alleles with more repetitions in NOS-1. However, alleles with minor repetitions in NOS-1 and CFTR showed association with the race and age. For race, the ratio between Caucasian and non-Caucasian was not similar and the p-value becomes irrelevant. Regarding age, only the CFTR was associated. Finally, there was association of minor repetitions to the TG1 and TG2 pol. in NOS-1, along with CFTR, with BS (measures the degree of impairment of structure bronchopulmonary) (Ratio: 0.954; Ball Test. Acc: 0.643, p: 0.033). The BS is a marker of early lung disease and is associated with the clinical severity, being considered an important factor in clinical prediction. Conclusion: NOS-1 and CFTR interaction influence BS value in CF. WS20.6 High throughput screening as a source for novel cystic fibrosis therapeutic targets H.M. Botelho1,2, S. Dahimene1,2, I. Uliyakina1,2, B. Neumann2, C. Tischer2, R. Pepperkok2, M.D. Amaral1. 1University of Lisboa, Faculty of Sciences (FCUL), BioFIG-Center for Biodiversity, Functional and Integrative Genomics, Lisboa, Portugal; 2European Molecular Biology Laboratory (EMBL), Cell Biology Biophysics Unit and Advanced Light Microscopy Facility (ALMF), Heidelberg, Germany