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The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

Galectins are soluble β-D-galactoside-binding proteins whose implication in cancer progression and disease outcome makes them prominent targets for therapeutic intervention. In this frame, the development of small inhibitors that block selectively the activity of galectins represents an important strategy for cancer therapy which is, however, still relatively underdeveloped. To this end, we designed here a rationally and efficiently novel diglycosylated compound, characterized by a selenoglycoside bond and the presence of a lipophilic benzyl group at both saccharide residues. The relatively high binding affinity of the new compound to the carbohydrate recognition domain of two galectins, galectin 3 and galectin 9, its good antiproliferative and anti-migration activity towards melanoma cells, as well as its anti-angiogenesis properties, pave the way for its further development as an anticancer agent.

Medulloblastoma (MB) is an embryonal tumor of the cerebellum constituting ~ 20% of pediatric brain tumors. To date, four MB molecular groups (further stratified in twelve subtypes) have been described. Among them, Groups 3 and Group 4 MB have the poorest prognosis due to their high metastatic potential. Recently, we have reported a metastatic axis driven by Prune1 overexpression in MB Group3 characterized by canonical TGF-β signaling enhancement and epithelial-mesenchymal transition. Here, we have developed a new not toxic pyrido-pyrimidine derivative with the ability to impair Prune-1-driven-axis, thus ameliorating the survival rate of a murine model of metastatic MB Group3 characterized by overexpression of human Prune1 gene in the cerebellum (under the control of MATH1 promoter). Of importance, this small molecule also is showing immunomodulatory functions thus inhibiting the conversion of tumor-infiltrating T lymphocytes (TILs) to immunosuppressive regulatory T cells (Tregs) in vivo via impairing the secretion of inflammatory cytokines from MB cells. Furthermore, this molecule can also act synergistically with the currently used modified-intensity chemotherapy (e.g. in PNET5 use of Vincristine) or potential in the combination with epigenetics drugs (e.g., LSD1/KDM1A inhibitors). Altogether these results are of importance for future targeted therapies of high-risk metastatic MB. Acknowledgments: We thank for funding support: the Italian Association for Cancer Research (AIRC) Grant IG no. 22129 (M.Z.) and Lazio Innova Grant n. 85-2017-14785 (FT; MZ)

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders., OFD1 (oro‐facial digital type I syndrome protein) resides in a centrosomal complex that links GPCR signalling to ubiquitylation and degradation of OFD1, controlling cilium morphology and dynamics and vertebrate development.

Galectins (Gals) are small cytosolic proteins that bind β-galactoside residues via their evolutionarily conserved carbohydrate recognition domain. Their dysregulation has been shown to be associated with many diseases. Consequently, targeting galectins for clinical applications has become increasingly relevant to develop tailored inhibitors selectively for one galectin. Accordingly, binding studies providing the molecular details of the interaction between galectin and inhibitor may be useful for the rational design of potent and selective antagonists. Gal-1 and Gal-3 are among the best-studied galectins, mainly for their roles in cancer progression; therefore, the molecular details of their interaction with inhibitors are demanded. This work gains more value by focusing on the interaction between Gal-1 and Gal-3 with the selenylated analogue of the Gal inhibitor thiodigalactose, characterized by a selenoglycoside bond (SeDG), and with unsymmetrical diglycosyl selenides (unsym(Se). Gal-1 and Gal-3 were produced heterologously and biophysically characterized. Interaction studies were performed by ITC, NMR spectroscopy, and MD simulation, and thermodynamic values were discussed and integrated with spectroscopic and computational results. The 3D complexes involving SeDG when interacting with Gal-1 and Gal-3 were depicted. Overall, the collected results will help identify hot spots for the design of new, better performing, and more specific Gal inhibitors.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported “KRI3” peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes, and triterpenes, which potentially have antiviral activities. As a consequence of the coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, thousands of scientists have been working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal of discovering effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. Using in vitro approaches, we investigated the role of a pomegranate peel extract (PPE) in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human angiotensin-converting enzyme 2 receptor, and on the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results opened new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2.

Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes and triterpenes, with potential antiviral activities. As the dramatic outbreak of the pandemic COVID-19, caused by the SARS-CoV-2 virus, thousands of scientists are working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal to discover effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. In this study, by using different in vitro approaches, we uncovered the role of a pomegranate peel extract in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, and in inhibiting the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results open up new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2.

Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56–142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-‘like’ gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1–55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein. The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein.

The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

The scalp-ear-nipple (SEN) syndrome is an autosomal-dominant disorder characterized by cutis aplasia of the scalp and malformations of breast, external ears, digits, and nails. Genetic analyses have shown that the disease is caused by missense mutations of the KCTD1 protein, although the functional/structural basis of SEN insurgence is hitherto unknown. With the aim of unravelling the molecular basis of the SEN syndrome associated with KCTD1 mutations we here expressed and characterized several disease causing mutants. A preliminary dissection of the protein provides insights into the role that individual domains play in KCTD1 stability. The characterization of SEN-causing mutants indicates that, although the mutation sites are located in distant regions of the BTB domain or of the pre-BTB region, all of them are unable to interact with the transcription factor AP-2α, a well-known KCTD1 biological partner. Notably, all mutations, including the one located in the pre-BTB region, produce a significant destabilization of the protein. The structural role of the pre-BTB region in KCTD1 and other proteins of the family is corroborated by its sequence conservation in orthologs and paralogs. Interestingly, SEN-causing mutations also favor the tendency of KCTD1 to adopt structural states that are characterized by the ability to bind the β-amyloid fluorescent dye thioflavin T. The formation of aggregation-prone species may have important implications for the disease etiology. Collectively, these findings provide an intriguing picture of the functional and structural alterations induced by KCTD1 mutations that ultimately lead to disease.

The transcription factors of the ArsR/SmtB family are widespread within the bacterial and archaeal kingdoms. They are transcriptional repressors able to sense a variety of metals and undergo allosteric conformational changes upon metal binding, resulting in derepression of genes involved in detoxification. So far, the molecular determinants of specificity, selectivity, and metal binding mechanism have been scarcely investigated in thermophilic microorganisms. TtSmtB, the only ArsR/SmtB member present in the genome of Thermus thermophilus HB27, was chosen as a model to shed light into such molecular mechanisms at high temperature. In the present study, using a multidisciplinary approach, a structural and functional characterization of the protein was performed focusing on its metal interaction and chemical-physical stability. Our data demonstrate that TtSmtB has two distinct metal binding sites per monomer and interacts with di-tri-penta-valent ions with different affinity. Detailed knowledge at molecular level of protein-metal interaction is remarkable to design metal binding domains as scaffolds in metal-based therapies as well as in metal biorecovery or biosensing in the environment.

Although the approved vaccines are proving to be of utmost importance in containing the Coronavirus disease 2019 (COVID-19) threat, they will hardly be resolutive as new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, a single-stranded RNA virus) variants might be insensitive to the immune response they induce. In this scenario, developing an effective therapy is still a dire need. Different targets for therapeutic antibodies and diagnostics have been identified, among which the SARS-CoV-2 spike (S) glycoprotein, particularly its receptor-binding domain, has been defined as crucial. In this context, we aim to focus attention also on the role played by the S N-terminal domain (S1-NTD) in the virus attachment, already recognized as a valuable target for neutralizing antibodies, in particular, building on a cavity mapping indicating the presence of two druggable pockets and on the recent literature hypothesizing the presence of a ganglioside-binding domain. In this perspective, we aim at proposing S1-NTD as a putative target for designing small molecules hopefully able to hamper the SARS-CoV-2 attachment to host cells.

The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam-Sam interactions involving EphA2.

The human flavoenzyme D-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator D-serine in the brain. Whereas hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C-terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG7272-153 form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in D-serine metabolism. This article is protected by copyright. All rights reserved.

Seven families of double-stranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and therefore represent model systems for detailed studies of archaeal virus biology. Two distinct genetic elements, SSV2 and pSSVx, belong to Fuselloviridae and coexist in the same Sulfolobus islandicus REY15/4 host, thus representing one of the few known two-virus systems in Archaea (1). pSSVx is a satellite virus that generates virus particles with the help of SSV2-associated packaging mechanisms. The transcriptional pattern of pSSVx undergoes a temporal variation of gene expression during its own life cycle, thus providing a good model for studying regulation of gene expression in Archaea (2). This genetic element encodes four TFs possibly implicated in the regulation of gene expression, i.e. ORF-c68, ORF51, ORF91 and ORF76. Among these, ORF76, here named Stf76 (Sulfolobus transcription factor 76 aminoacid protein), has homologs in almost all conjugative and cryptic plasmids from Sulfolobus (3), thus suggesting a relevant role for this protein in replication and/or maintenance of the plasmid. In this study, we have performed a detailed structural and functional characterization of Stf76. The corresponding gene has been cloned, expressed in Escherichia coli and the recombinant protein purified to homogeneity. To elucidate its interaction with the identified DNA operator sequence, analyses regarding its DNA binding capabilities by means of EMSA, circular dichroism, spectrofluorimetric and isothermal titration calorimetry experiments have been performed. Moreover, a structural study has been undertaken by Nuclear Magnetic Resonance spectroscopy leading to:(i) the solution structure of Stf76 based on CS-Rosetta approach, (ii) the characterization of the Stf76-DNA interaction by chemical shift perturbation analysis, (iii) a structural model describing the interaction of a single Stf76 monomer with its DNA operator. Altogether these results contribute to elucidate the regulatory mechanism underpinning the role of this protein (4). REFERENCES: 1. ARNOLD, H.P., ET AL.(1999) THE GENETIC ELEMENT PSSVX OF THE EXTREMELY THERMOPHILIC CRENARCHAEON SULFOLOBUS IS A HYBRID BETWEEN A PLASMID AND A VIRUS. MOL MICROBIOL, 34, 217-226. 2. CONTURSI, P., ET.AL. (2010) TRANSCRIPTION TERMINATION IN THE PLASMID/VIRUS HYBRID PSSVX FROM SULFOLOBUS ISLANDICUS. EXTREMOPHILES, 14, 453-463. 3. LIPPS, G. (2006) PLASMIDS AND VIRUSES OF THE THERMOACIDOPHILIC CRENARCHAEOTE SULFOLOBUS. EXTREMOPHILES, 10, 17-28. 4. CONTURSI P., FARINA B., PIRONE L. ET AL. NUCLEIC ACIDS RES. ACCEPTED 28 FEB. 2014.

In this paper, we report studies concerning thrombin binding aptamer (TBA) dimeric derivatives in which the 3′-ends of two TBA sequences have been joined by means of linkers containing adenosine or thymidine residues and/or a glycerol moiety. CD and electrophoretic investigations indicate that all modified aptamers are able to form G-quadruplex domains resembling that of the parent TBA structure. However, isothermal titration calorimetry measurements of the aptamer/thrombin interaction point to different affinities to the target protein, depending on the type of linker. Consistently, the best ligands for thrombin show anticoagulant activities higher than TBA. Interestingly, two dimeric aptamers with the most promising properties also show far higher resistances in biological environment than TBA.

Appetite for ubiquitin: a gushy travel companion in the intracellular journey of the amyloid β peptide., Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid β peptide (Aβ) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aβ peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aβ40 binds Ub with a 1 : 1 stoichiometry and Kd in the high micromolar range. In particular, we show that the N-terminal domain of the Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/β-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1–16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aβ peptide in the malfunction of proteome maintenance occurring in AD.

Carnosine, anserine and homocarnosine are the three most representative compounds of the histidine dipeptides family, widely distributed in mammals in different amounts depending on the species and the tissue considered. Histidine dipeptides are mainly degraded by two different carnosinase homologues: a highly specific metal-ion dependent carnosinase (CN1) located in serum and brain and a non-specific cytosolic form (CN2). The hydrolysis of such dipeptides in prokaryotes and eukaryotes is also catalyzed by the anserinase (ANSN). Such naturally occurring dipeptides represent an interesting topic because they seem to have numerous biological roles such as potential neuroprotective and neurotransmitter functions in the brain and therefore ANSN results to be a very interesting target of study. We here report, for the first time, cloning, expression of ANSN from the fish Oreochromis niloticus both in a mammalian and in a prokaryotic system, in order to perform deep functional studies by enzymatic assays in the presence of different metals and substrates. Furthermore, by means of a mass spectrometry-based proteomic approach, we analysed protein sequence and the potential presence of post-translational modifications in the mammalian recombinant protein. Finally, a preliminary structural characterization was carried out on ANSN produced in Escherichia coli.

A mini-library of symmetrical and unsymmetrical diglycosyl (di)sulfides, containing D-galactose, L-fucose and N-acetyl glucosamine units, were synthesized and tested for the antiproliferative activity against cervix carcinoma (HeLa) and melanoma (A375) tumor cell lines as well as healthy fibroblasts (HDF). Comparative analysis of results seems to indicate that the most relevant antiproliferative effect is not primarily influenced by interactions with galectins, as the most cytotoxic compound observed for HeLa and A375 is not a ligand for such receptors. The most active molecules against HeLa and A375 lines also exhibited a good selectivity, showing a low toxicity to HDF cells. Obtained results offer useful indications for future design of structurally simple antitumor molecules based on sugar moieties with bridging sulfur atoms.

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.

Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam.

Protein disulfide oxidoreductases (PDOs) are proteins involved in disulfide bond formation playing a crucial role in adaptation to extreme environment. This paper reports the functional and structural characterization of Sso1120, a PDO from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was expressed in Escherichia coli and purified to homogeneity. The functional characterization showed that the enzyme has reductase activity, as tested by insulin assay, but differently from the other PDOs, it does not present isomerase activity. In addition it is able to form a redox couple with the thioredoxin reductase that could be used in undiscovered pathways. The protein revealed a melting point of around 90 °C in CD spectroscopy-monitored thermal denaturation and high denaturant resistance. The X-ray crystallographic structure was solved at 1.80 Å resolution, showing differences with respect to other PDOs and an unexpected similarity with the N-terminal domain of the alkyl hydroperoxide reductase F component from Salmonella typhimurium. On the basis of the reported data and of bioinformatics and phylogenetic analyses, a possible involvement of this atypical PDO in a new antioxidant system of S. solfataricus has been proposed. © 2013 Springer Japan.

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions.

KCTD15 is a member of the K+ Channel Tetramerization Domain family, implicated in crucial physio-pathological processes. Recent evidences suggest that KCTD15 is an obesity-linked protein in humans and its Drosophila homologue is involved in food uptake. KCTD15 molecular mechanism in these processes is still unknown. To fill this gap, KCTD15 was biophysically characterized showing a folded, pentameric region endowed with a remarkable thermal stability. Notably, the C-terminal domain significantly contributes to the stabilization of the BTB N-terminal domain. The availability of large amount of stable recombinant protein also made possible a functional proteomic approach in 3T3-L1 cells to search for novel KCTD15 interactors. These investigations led to the discovery that GRP78 is a KCTD15 partner in all the adipogenesis phases. Our data clearly prove the physical interaction of the two proteins and also indicate that GRP78 plays an active role in the stabilization of KCTD15. Furthermore, the presence in Drosophila of a GRP78 homologue corroborates the physiological role played by the complex KCTD15-GRP78 in the adipogenesis process and indicates that it is evolutionarily conserved. Present results also suggest that KCTD15 may be a new target for obesity control.

A RGD motif was identified in the N-terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit ?? and ?? mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiae species complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward ??. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA-MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent.

Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped>flow, T>jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, the two zinc binding Ros87, Ml1 53>149 and the zinc>lacking Ml4 52−151 . The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml1 53>149 appear much more stable at guanidinium denaturation and characterized by folding mechanisms including the presence of an intermediate. On the other hand, the metal lacking Ml4 52 −151 folds according to a classic two>state model. Successively, we have monitored Ros87, Ml4 52 −151 and Ml1 53>149 capabilities to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours Ros87 amyloid formation has started, while Ml1 53>149 has formed only amorphous aggregates and Ml4 52 −151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation>prone states, which in turn changes aggregation kinetics, sheding light onto the role of metal ions in the development of protein deposition diseases. Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml153-149 and zinc-lacking Ml452-151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153-149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml452-151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452-151 and Ml153-149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml153-149 has formed only amorphous aggregates and Ml452-151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition diseases.

Background The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. Methods Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. Results It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. Conclusions VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. General significance VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Saliva of blood-feeding arthropods carries several antihemostatic compounds whose physiological role is to facilitate successful acquisition of blood. The identification of novel natural anticoagulants and the understanding of their mechanism of action may offer opportunities for designing new antithrombotics disrupting blood clotting. We report here an in-depth structural and functional analysis of the anophelin family member cE5, a salivary protein from the major African malaria vector Anopheles gambiae that specifically, tightly, and quickly binds and inhibits thrombin. Using calorimetry, functional assays, and complementary structural techniques, we show that the central region of the protein, encompassing amino acids Asp-31-Arg-62, is the region mainly responsible for α-thrombin binding and inhibition. As previously reported for the Anopheles albimanus orthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic site with the region Pro-49-Arg-56, which includes the highly conserved DPGR tetrapeptide. Moreover, the N-terminal Ala-1-Ser-30 region of cE5 (which includes an RGD tripeptide) and the additional C-terminal serine-rich Asn-63-Glu-82 region (absent in orthologues from anophelines of the New World species A. albimanus and Anopheles darlingi) also played some functionally relevant role. Indeed, we observed decreased thrombin binding and inhibitory properties even when using the central cE5 fragment (Asp-31-Arg-62) alone. In summary, these results shed additional light on the mechanism of thrombin binding and inhibition by this family of salivary anticoagulants from anopheline mosquitoes.

Adipogenesis has an important role in regulating energy balance, tissue homeostasis and disease pathogenesis. 3T3-L1 preadipocytes have been widely used as an in vitro model for studying adipocyte differentiation. We here show that KCTD1, a member of the potassium channel containing tetramerization domain proteins, plays an active role in adipogenesis. In particular, we show KCTD1 expression 3T3-L1 cells increases upon adipogenesis induction. Treatment of 3T3-L1 preadipocytes with Kctd1-specific siRNA inhibited the differentiation, as indicated by reduction of expression of the specific adipogenic markers C/ebpa, Ppar gamma 2, Glut4, and Adiponectin. Moreover, we also show that the protein physically interacts with the transcription factor AP2 alpha, a known inhibitor of adipogenesis, both in vitro and in cells. Interestingly, our data indicate that KCTD1 promotes adipogenesis through the interaction with AP2 alpha a and by removing it from the nucleus. Collectively, these findings disclose a novel role for KCTD1 and pave the way for novel strategies aimed at modulating adipogenesis.

Recent investigations have shown that members of the KCTD family play important roles in fundamental biological processes. Despite their roles, very limited information is available on their structures and molecular organization. By combining different experimental and theoretical techniques, we have here characterized the two folded domains of KCTD12, an integral component and modulator of the GABAB2 receptor. Secondary prediction methods and CD spectroscopy have shown that the N-terminal domain KCTD12BTB assumes an α/β structure, whereas the C-terminal domain KCTD12H1 is predominantly characterized by a β-structure. Binding assays indicate that the two domains independently expressed show a good affinity for each other. This suggests that the overall protein is likely endowed with a rather compact structure with two interacting structured domains joint by a long disordered region. Notably, both KCTD12BTB and KCTD12H1 are tetrameric when individually expressed. This finding could modify the traditional view that ascribes only to POZ/BTB domain a specific oligomerization role. The first quantification of the affinity of KCTD12POZ/BTB for the C-terminal region of GABAB2 shows that it falls in the low micromolar range. Interestingly, we also demonstrate that a GABAB2 -related peptide is able to bind KCTD12BTB with a very high affinity. This peptide may represent a useful tool for modulating KCTD12/GABAB2 interaction in vitro and may also constitute the starting point for the development of peptidomimetic compounds with a potential for therapeutic applications.

In the human brain, pLG72 interacts with the flavoenzyme d-amino acid oxidase (hDAAO), which is involved in catabolism of d-serine, a co-agonist of N-methyl-d-aspartate receptors (NMDAR). Here, we investigated the wild-type pLG72, the R30K variant associated with schizophrenia susceptibility, and the K62E variant. The protein conformation, oligomeric state, ligand-, and hDAAO-binding properties are only slightly modified by the substitutions. All pLG72 variants inhibit hDAAO and lead to an increase in cellular (d/d+l)-serine. However, the R30K pLG72 is significantly more prone to degradation than the R30 and the K62E variants in a cell system, thus possessing a lower ability to interact/inhibit hDAAO. This links R30K pLG72 with the hyperactivity of hDAAO, the decreased d-serine level, and NMDAR hypofunction observed in schizophrenia-affected patients.

Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium lo ti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization. Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium loti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an alpha-helix and two short beta-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains.

POZ/BTB domains are widespread modules detected in a variety of different biological contexts. Here, we report a biophysical characterization of the POZ/BTB of KCTD6, a protein that is involved in the turnover of the muscle small ankyrin-1 isoform 5 and, in combination with KCTD11, in the ubiquitination and degradation of HDAC1. The analyses show that the domain is a tetramer made up by subunits with the expected ?/? structure. A detailed investigation of its stability, carried out in comparison with the homologous pentameric POZ/BTB domain isolated from KCTD5, highlights a number of interesting features, which are shared by the two domains despite their different organization. Their thermal/chemical denaturation curves are characterized by a single and sharp inflection point, suggesting that the denaturation of the two domains is a cooperative two-state process. Furthermore, both domains present a significant content of secondary structure in their denatured state and a reversible denaturation process. We suggest that the ability of these domains to fold and unfold reversibly, a property that is somewhat unexpected for these oligomeric assemblies, may have important implications for their biological function. Indeed, these properties likely favor the formation of heteromeric associations that may be essential for the intricate regulation of the processes in which these proteins are involved. © 2013 Luciano Pirone et al.

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2-receptor (EphA2-Sam). Both Arap3-Sam and EphA2-Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2-Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin-Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2-Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3-Sam and Odin-Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin-Sam1 and Arap3-Sam might bind with a topology that is common to several Sam-Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3-Sam associations.

Pox virus Zinc/Bric-a-brac, Tramtrack and Broad (POZ/BTB) is a widespread domain detected in proteins involved in a variety of biological processes. Human genome analyses have unveiled the presence of POZ/BTB domain in a class of proteins (KCTD) whose role as important players in crucial biological processes is emerging. The development of new molecular entities able to interact with these proteins and to modulate their activity is a field of relevant interest. By using molecular modeling and literature mutagenesis analyses, we here designed and characterized a peptide that is able to interact with submicromolar affinities with two different members (KCTD11 and KCTD5) of this family. This finding suggests that the tetrameric KCTD11 and the pentameric KCTD5 are endowed with a similar cavity at the subunit-subunit interface deputed to the Cul3 binding, despite their different oligomeric states.