Prediction of proprotein convertase cleavage sites

Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.     

Targeted Delivery of an Activatable Fluorescent Probe for the Detection of Furin Activity in Living Cells

Furin, a protein convertase, plays a crucial role in the progression of tumors. In this work, a new fluorescent probe consisting of a peptide, Arg‐Val‐Arg‐Arg (RVRR), and an aminoluciferin fluorophore was designed and prepared for the responsive and activatable detection of furin activity in vitro and in living cells. We demonstrated that this probe could be responsive toward furin with an “off–on” florescence signal and generated an approximately 3.58‐fold enhancement in the fluorescence intensity in vitro. Fluorescence imaging in MDA‐MB‐468 and LoVo cells showed that the probe could be cleaved by overexpressed furin with fluorescence turn‐on in MDA‐MB‐468 cells, and this probe was also found to be capable of discriminating between furin‐overexpressing and furin‐deficient cell lines. Our research indicates that this probe has great potential for the detection of furin activity in living cells.     

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site, 2: relevance of an N-terminal helix

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.     

Semisynthesis and characterization of the first analogues of pro-neuropeptide y

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs

FurinDB (freely available online at http://www.nuolan.net/substrates.html) is a database of furin substrates. This database includes experimentally verified furin cleavage sites, substrates, species, experimental methods, original publications of experiments and associated drugs targeting furin substrates. The current database release contains 126 furin cleavage sites from three species: mammals, bacteria and viruses. A main feature of this database is that all furin cleavage sites are recorded as a 20-residue motif, including one core region (eight amino acids, P6-P2') and two flanking solvent accessible regions (eight amino acids, P7-P14, and four amino acids, P3'-P6'), that represent our current understanding of the molecular biology of furin cleavage. This database is important for understanding the molecular evolution and relationships between sequence motifs, 3D structures, cellular functions and physical properties required by furin for cleavage, and for elucidating the molecular mechanisms and the progression of furin cleavage associated human diseases, including pathogenic infections, neurological disorders, tumorigenesis, tumor invasion, angiogenesis, and metastasis. FurinDB database will be a solid addition to the publicly available infrastructure for scientists in the field of molecular biology.     

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.     

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site 3: role of site-specific mutations

Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date.In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.     

Preparation of amphoteric polycarboxylate superplasticizers and their performances in cementitious system

A series of amphoteric polycarboxylate (PC) polymers were synthesized by radical copolymerization of acrylic acid (AA), [3‐(methacryloylamino) propyl] trimethylammonium chloride (MAPTAC) and ω‐methoxypolyoxyethylene methacrylate ester (MPEGMA). Cationic groups were introduced in to PC molecules with expectation of less retardation effect on cement hydration compared to the traditional anionic PC superplasticizers. The content of cationic groups in polymer was varied by changing the monomer ratio of MAPTAC to AA in the synthesis recipes. The structure of the synthesized amphoteric PCs was verified by gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FTIR). The performances of the amphoteric PCs were evaluated by measurement of flowability and zeta‐potential of cement pastes and adsorption amount of PC in cement pastes. Impacts of the PCs on cement hydration were studied by isothermal calorimetry. It is concluded that both anionic and cationic PC polymers can be effectively adsorbed onto the surface of cement particles and thus change the zeta potential of cement pastes. The adsorption amounts of the amphoteric PCs decrease with increasing content of cationic units. A proper incorporation of cationic units into PC polymers may lead to a higher fluidizing performance in fresh cement pastes. The amphoteric PC polymers with higher content of cationic units show less retardation effect on cement hydration and hence higher early strength of cementitious materials may be achieved by using amphoteric PCs with appropriated content of cationic units without losing their plasticizing efficiency. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41348.     

Ionic pseudopolyrotaxanes bearing a chromophore in the side chain - A spectroscopic study in water

The formation of pseudopolyrotaxanes based on a polyelectrolyte, a surfactant and α-cyclodextrin (α-CD) in aqueous solution is investigated. The polyelectrolyte and the surfactant form an ionic complex the side chains of which interact with each other. These interactions are strongly influenced by the formation of inclusion complexes with α-CD. It is shown by ¹H NMR and UV-vis spectroscopy that threading of the α-CD rings onto the side chains causes a shielding effect which increases the mobility of the side chains. The structure of the pseudopolyrotaxanes is concluded from ROESY spectra and their stoichiometry is determined by Job plots based on shift effects observed in the ¹H NMR spectra.     

Structural Rearrangement from Oligomer to Fibril Detected with FRET in a Designed Amphiphilic Peptide

β‐Sheet conformation is promoted in peptides with amphiphilic design, and stable β‐turn formation is favored with the unnatural amino acid d ‐Pro followed by a flexible residue such as Gly. A 19‐residue peptide (B3) was synthesized with alternating hydrophobic and hydrophilic residues connected by symmetrical d ‐Pro‐Gly and Gly‐d ‐Pro turns. B3 forms an oligomeric aggregate, rich in β‐sheet conformation, that reversibly transforms into an unordered structure on heating, as evidenced by its temperature‐dependent IR spectra. When a dansyl moiety was added to the N terminus of B3, the resulting peptide (B3D) can convert into a fibrillar structure after higher temperature incubation, as detected spectroscopically as well as by TEM. The fibrillization process involves an initial unfolding step monitored by the quenching of dansyl emission; in contrast, subsequent enhanced thioflavin T emission is seen on its binding to the fibril. A possible mechanism is proposed: B3D forms a low‐temperature oligomer, which is at least partially unfolded by heat and subsequently reassembles more slowly as a fibril.     

NMR and IR study of kaolinite intercalation compounds with benzylalkylammonium chlorides

Novel intercalation compounds of kaolinite with selected benzylalkylammonium chlorides, obtained using methoxy-kaolinite as a precursor, were characterized by ¹³C, ²⁷Al, and ²⁹Si solid-state NMR and infrared (FTIR) spectroscopy. The analyses allowed the confirmation of both the formation and structure of precursors (kaolinite-dimethyl sulfoxide and methoxy-kaolinite), as well as the intercalation compounds. According to the ¹³C CP-MAS NMR spectra of the derivatives with benzyltrimethylammonium chloride (B1), the methyl groups of the molecules, arranged in a monolayer, were keyed into the ditrigonal holes of the kaolinite tetrahedral sheet. The local environment of internal carbons of benzyltributylammonium chloride (B2) butyl chains was mostly perturbed during intercalation. In turn, the structure of the benzyldimethylhexadecylammonium chloride (B5) after intercalation was changed from the solid-like to liquid-like conformation with an increase of gauche conformers, which was attested both by ¹³C CP-MAS NMR and FTIR. The ¹³C CP-MAS NMR analysis confirmed that the methoxyl groups attached to the octahedral surface of methoxy-kaolinite were not affected by intercalation. The ²⁷Al and ²⁹Si MAS NMR did not show any significant distortion of the Al-octahedra and Si-tetrahedra after intercalation of the organic salts. The IR analyses indicated an increased amount of intercalated water with the increase of initial salt concentration.     

Adhesion failure of antiscratch coatings on polycarbonate under UV irradiation

The adhesion failure of antiscratch (AS) coatings on unmodified and plasma‐modified polycarbonate (PC) substrate was studied using both chemical and physical methods while considering the surface and interface changes between coatings and PC under ultraviolet (UV) irradiation. The differences in the wettability and surface elemental compositions of the PC surface (PCs) and AS coatings after UV ageing were evaluated by contact angle and Fourier transform infrared (FTIR) measurements. The nanoindentation technique was employed for the quantitative assessment of the changes in the nanomechanical properties of both PCs and AS coatings under UV irradiation. The adhesion of coating on plasma‐modified PC was found to be significantly better than that of unmodified substrates. The hydrophilicity and polarity of PCs covered by AS coatings were significantly increased because of the photodegradation of PCs, whereas silicon coatings remained invariant. Nanoindentation tests revealed an obvious enhancement in stiffness of the coating and exposed PC after ageing. Based on these experiments, we proposed that adhesion failure under UV irradiation may be caused by two reasons: first, the photodegradation that occurred at the PCs covered by AS coatings; and second, the stress induced by the changes in stiffness of both AS and PCs under UV ageing. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40507.     

Preparation and characterization of porous carbons from PAN-based preoxidized cloth by KOH activation

Porous carbons (PCs) were prepared from PAN-based preoxidized cloth with potassium hydroxide (KOH) as active reagent by the chemical activation method. The PCs have been systematically studied by the adsorptions of nitrogen, benzene and iodine. It has been found that the process parameters such as weight ratio of KOH to the starting material, activation temperature and activation time are crucial for preparing high quality PCs. A series of PCs with high BET surface area and well-developed porous structure in which micropores are dominant were obtained with less KOH and shorter activation time in comparison to the traditional methods. The optimum conditions for preparing PCs with high BET surface area from PAN-based preoxidized cloth were given, and the relationships between pore structure and adsorption property of PCs were explored.     

Fullerene‐functionalized polycarbonate: Synthesis under microwave irradiation and nonlinear optical property

Fullerenation of polycarbonate (PC), a commercially important optical polymer, was achieved by direct reaction of C₆₀ and PC in the presence of azo‐bis‐isobutyronitrile (AIBN), using 1,1,2,2‐tetrachloroethane as the solvent under microwave irradiation (MI). Compared with conventional heating process, MI could significantly enhance the rate of the fullerenation under identical reaction conditions. The C₆₀ content of the fullerene‐functionalized polycarbonate (C₆₀‐PC) could be controlled via varying the C₆₀/PC feed ratio and the reaction time. The C₆₀‐PCs are soluble in common organic solvents such as THF and chloroform. The products were characterized by gel permeation chromatography, UV–vis, FTIR, TGA, DSC, ¹H NMR, and ¹³C NMR. The reaction of C₆₀ with PC under MI was monitored by electron spin resonance spectra, the fullerene radicals were detected in reaction solutions and also in the solid product polymers, indicating the radical mechanism of the reaction. The nonlinear optical property of C₆₀‐PCs in THF was investigated by the open‐aperture z‐scan technique at 527 nm, and its nonlinear absorption coefficient was found to be in the same order as that of C₆₀. POLYM. ENG. SCI., 46:399–405, 2006. © 2006 Society of Plastics Engineers     

Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3

We have engineered a latent mouse interferon beta (mIFNbeta) using the latency associated peptide (LAP) of transforming growth factor beta1 (TGF-beta1) to protect the cytokine and avoid its interaction with its receptors. This approach improves the pharmacokinetic properties and reduces the pleiotropic effects limiting the current therapeutic use of cytokines. IFNbeta was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNbeta at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFNbeta construct. The kinetic parameters relative to the linker cleavage by MMP-1 and MMP-3 were measured by an ELISA method. Among the modifications done, one of the constructs bearing the activation site of pro-MMPs was the best substrate for both enzymes. The introduction of a hydrophilic sequence derived from the furin cleavage site of the anthrax toxin protective antigen increased the sensitivity to MMP-3 to up to 29-fold. These data suggest that this strategy could be useful for improving the effectiveness of the delivery and targeting of protein therapeutics.     

Crystal structure of a synthetic cyclodecapeptide for template-assembled synthetic protein design

The structural prototype of a new generation of regioselectively addressable functionalized templates (RAFTs) for use in protein de novo design has been synthesized and crystallized. The structure of the aromatically substituted cyclodecapeptide was determined by X-ray diffraction; it consists of an antiparallel beta sheet spanned by heterochirally induced type IIprime prime or minute beta turns, similar to that observed in gramicidin S. The three-dimensional structure of the artificial template was also examined by an NMR spectroscopic analysis in solution and shown to be compatible with a beta-sheet plane suitable for accommodating secondary functional peptide fragments for the synthesis of template-assembled synthetic proteins (TASPs).     

Identification of Kukoamine A,Zeaxanthin, and Clexane as New Furin Inhibitors

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.     

Novel Furin Inhibitors with Potent Anti‐infectious Activity

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4‐guanidinomethyl‐phenylacteyl‐Arg‐Tle‐Arg‐4‐amidinobenzylamide (MI‐1148) inhibits furin with a K_i value of 5.5 pM . The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI‐1148 was crystallized in complex with human furin. Its N‐terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short‐term treatment of acute infectious diseases.     

Engineering of alpha1-antitrypsin variants selective for subtilisin-like proprotein convertases PACE4 and PC6: importance of the P2' residue in stable complex formation of the serpin with proprotein convertase

Furin and PACE4, members of the subtilisin-like proprotein convertase (SPC) family, have been implicated in the metastatic progression of certain tumors in addition to the activation of viral coat proteins and bacterial toxins, indicating that these enzymes are potential targets for therapeutic agents. Alpha1-Antitrypsin Portland is an engineered alpha1-antitrypsin designed as a furin-specific inhibitor and has been used as a tool in the functional analysis of furin. In this work, we engineered rat alpha1-antitrypsin to create a PACE4-specific inhibitor. Substituting Arg-Arg-Arg-Arg for Ala-Val-Pro-Met(352) at P4-P1 and Ala for Leu(354) at P2' created a potent PACE4- and PC6-specific inhibitor. This variant (RRRRSA) formed an SDS- and heat-stable serpin/proteinase complex with PACE4 or PC6 and inhibited both enzyme activities. The RRRRSA variant was efficiently cleaved by furin without formation of the stable complex. This is the first report of a highly selective protein-based inhibitor of PACE4 and PC6. This inhibitor will be useful in delineating the roles of PACE4 and PC6 localized in the extracellular matrix.