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1.
J Am Chem Soc ; 143(42): 17615-17621, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34647745

RESUMO

Cellular binding and entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are mediated by its spike glycoprotein (S protein), which binds with not only the human angiotensin-converting enzyme 2 (ACE2) receptor but also glycosaminoglycans such as heparin. Cell membrane-coated nanoparticles ("cellular nanosponges") mimic the host cells to attract and neutralize SARS-CoV-2 through natural cellular receptors, leading to a broad-spectrum antiviral strategy. Herein, we show that increasing surface heparin density on the cellular nanosponges can promote their inhibition against SARS-CoV-2. Specifically, cellular nanosponges are made with azido-expressing host cell membranes followed by conjugating heparin to the nanosponge surfaces. Cellular nanosponges with a higher heparin density have a larger binding capacity with viral S proteins and a significantly higher inhibition efficacy against SARS-CoV-2 infectivity. Overall, surface glycan engineering of host-mimicking cellular nanosponges is a facile method to enhance SARS-CoV-2 inhibition. This approach can be readily generalized to promote the inhibition of other glycan-dependent viruses.


Assuntos
COVID-19/tratamento farmacológico , Heparina/administração & dosagem , Nanoestruturas/uso terapêutico , Polissacarídeos/administração & dosagem , SARS-CoV-2/metabolismo , COVID-19/virologia , Heparina/metabolismo , Humanos , Polissacarídeos/metabolismo
2.
Elife ; 102021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477104

RESUMO

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).


Assuntos
Amina Oxidase (contendo Cobre) , Motivos de Aminoácidos/genética , Produtos Biológicos , Heparina/metabolismo , Antagonistas dos Receptores Histamínicos , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Antagonistas dos Receptores Histamínicos/química , Antagonistas dos Receptores Histamínicos/metabolismo , Humanos , Camundongos , Mutação/genética , Ligação Proteica/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Front Immunol ; 12: 676662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489931

RESUMO

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/sangue , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Células Cultivadas , Ativação do Complemento , Células Endoteliais/metabolismo , Heparina/análogos & derivados , Heparina/farmacologia , Humanos , Glomérulos Renais/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
4.
Mar Drugs ; 19(8)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34436245

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a novel coronavirus strain that emerged at the end of 2019, causing millions of deaths so far. Despite enormous efforts being made through various drug discovery campaigns, there is still a desperate need for treatments with high efficacy and selectivity. Recently, marine sulfated polysaccharides (MSPs) have earned significant attention and are widely examined against many viral infections. This article attempted to produce a comprehensive report about MSPs from different marine sources alongside their antiviral effects against various viral species covering the last 25 years of research articles. Additionally, these reported MSPs were subjected to molecular docking and dynamic simulation experiments to ascertain potential interactions with both the receptor-binding domain (RBD) of SARS CoV-2's spike protein (S-protein) and human angiotensin-converting enzyme-2 (ACE2). The possible binding sites on both S-protein's RBD and ACE2 were determined based on how they bind to heparin, which has been reported to exhibit significant antiviral activity against SARS CoV-2 through binding to RBD, preventing the virus from affecting ACE2. Moreover, our modeling results illustrate that heparin can also bind to and block ACE2, acting as a competitor and protective agent against SARS CoV-2 infection. Nine of the investigated MSPs candidates exhibited promising results, taking into consideration the newly emerged SARS CoV-2 variants, of which five were not previously reported to exert antiviral activity against SARS CoV-2, including sulfated galactofucan (1), sulfated polymannuroguluronate (SPMG) (2), sulfated mannan (3), sulfated heterorhamnan (8), and chondroitin sulfate E (CS-E) (9). These results shed light on the importance of sulfated polysaccharides as potential SARS-CoV-2 inhibitors.


Assuntos
Antivirais/farmacologia , Organismos Aquáticos/química , Polissacarídeos/farmacologia , SARS-CoV-2/química , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Sítios de Ligação , Simulação por Computador , Heparina/química , Heparina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Polissacarídeos/química , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade , Sulfatos/química
5.
J Virol ; 95(21): e0135721, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406867

RESUMO

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Assuntos
Evolução Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Biológica , Animais , Sítios de Ligação , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar , Furina/metabolismo , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral , Ligação Viral
6.
Sci Rep ; 11(1): 15579, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341408

RESUMO

Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.


Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Fator 1 de Crescimento de Fibroblastos/metabolismo , Desdobramento de Proteína , Temperatura , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Guanidina/farmacologia , Heparina/metabolismo , Humanos , Camundongos , Proteínas Mutantes/química , Mutação/genética , Células NIH 3T3 , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Estabilidade Proteica , Eletricidade Estática , Ureia/farmacologia
7.
Biomater Sci ; 9(16): 5612-5625, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34254062

RESUMO

This study reports that the use of low-frequency sonophoresis (LFS) in combination with sponge Haliclona sp. spicules (SHS), referred to as cSoSp (combined Sonophoresis and Spicules), can enhance the transdermal drug delivery in a synergistic manner. The topical application of cSoSp in vitro significantly enhanced the skin absorption of Fluorescent-Dextrans (4000 Da, FD-4K), a model drug of low-molecular-weight heparin (LMWH). The utilization of cSoSp dramatically increased the transdermal flux of FD-4K (188.6 ± 93.7 ng cm-2 h-1) compared to LFS (5.8 ± 3.1 ng cm-2 h-1) and SHS (3.2 ± 1.2 ng cm-2 h-1) among others. The mechanism of action of cSoSp could be attributed to the synergism between plenty of long-lasting nano-channels created by SHS and the disorders of SC lipids made by shock waves of LFS, which improves the homogeneity of the cavitation effects. Furthermore, LMWH (3000 Da) was transdermally delivered by using cSoSp to treat both superficial venous thrombosis (SVT) and deep venous thrombosis (DVT) in the marginal ear vein of rabbits with a good therapeutic effect. Furthermore, skin irritation and toxicity studies using guinea pigs indicated that cSoSp was nonirritating without any morphological changes in the keratinocytes. cSoSp offers a promising strategy to enhance the transdermal delivery of hydrophilic macromolecules such as heparin.


Assuntos
Heparina , Trombose Venosa , Administração Cutânea , Animais , Cobaias , Heparina/metabolismo , Heparina de Baixo Peso Molecular , Coelhos , Pele/metabolismo , Absorção Cutânea , Trombose Venosa/tratamento farmacológico , Trombose Venosa/metabolismo
8.
Nat Commun ; 12(1): 4074, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210960

RESUMO

The c-MET receptor is a receptor tyrosine kinase (RTK) that plays essential roles in normal cell development and motility. Aberrant activation of c-MET can lead to both tumors growth and metastatic progression of cancer cells. C-MET can be activated by either hepatocyte growth factor (HGF), or its natural isoform NK1. Here, we report the cryo-EM structures of c-MET/HGF and c-MET/NK1 complexes in the active state. The c-MET/HGF complex structure reveals that, by utilizing two distinct interfaces, one HGF molecule is sufficient to induce a specific dimerization mode of c-MET for receptor activation. The binding of heparin as well as a second HGF to the 2:1 c-MET:HGF complex further stabilize this active conformation. Distinct to HGF, NK1 forms a stable dimer, and bridges two c-METs in a symmetrical manner for activation. Collectively, our studies provide structural insights into the activation mechanisms of c-MET, and reveal how two isoforms of the same ligand use dramatically different mechanisms to activate the receptor.


Assuntos
Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Células HEK293 , Heparina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Receptores da Neurocinina-1/metabolismo
9.
Blood ; 138(14): 1269-1277, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280256

RESUMO

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.


Assuntos
Anticorpos/efeitos adversos , Vacinas contra COVID-19/efeitos adversos , Reações Cruzadas/imunologia , Fator Plaquetário 4/imunologia , Púrpura Trombocitopênica Idiopática/etiologia , Púrpura Trombocitopênica Idiopática/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/imunologia , COVID-19/imunologia , Estudos de Coortes , Epitopos/imunologia , Feminino , Heparina/metabolismo , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Domínios Proteicos , Púrpura Trombocitopênica Idiopática/sangue , Glicoproteína da Espícula de Coronavírus/química , Adulto Jovem
10.
Nature ; 596(7873): 565-569, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34233346

RESUMO

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor 4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n = 5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n = 10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptor IIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.


Assuntos
Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , Epitopos de Linfócito B/imunologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/imunologia , Trombose/induzido quimicamente , Trombose/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Feminino , Heparina/química , Heparina/imunologia , Heparina/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Ativação Plaquetária , Fator Plaquetário 4/imunologia , Receptores de IgG/imunologia
11.
J Gen Virol ; 102(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34106826

RESUMO

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.


Assuntos
Heparina/metabolismo , Sulfatos/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Penaeidae/virologia , Ligação Proteica , Conformação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética
13.
FASEB J ; 35(7): e21647, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165206

RESUMO

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfócitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/genética
14.
J Virol ; 95(18): e0059321, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160259

RESUMO

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.


Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Heparina/metabolismo , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Feminino , Heparina/análogos & derivados , Humanos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Paramyxoviridae/metabolismo , Infecções por Paramyxoviridae/virologia , Ligação Proteica , Conformação Proteica , Proteoglicanas/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Proteínas Virais de Fusão/metabolismo
15.
Biomolecules ; 11(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800172

RESUMO

The tumor microenvironment (TME) is composed of cancerous, non-cancerous, stromal, and immune cells that are surrounded by the components of the extracellular matrix (ECM). Glycosaminoglycans (GAGs), natural biomacromolecules, essential ECM, and cell membrane components are extensively altered in cancer tissues. During disease progression, the GAG fine structure changes in a manner associated with disease evolution. Thus, changes in the GAG sulfation pattern are immediately correlated to malignant transformation. Their molecular weight, distribution, composition, and fine modifications, including sulfation, exhibit distinct alterations during cancer development. GAGs and GAG-based molecules, due to their unique properties, are suggested as promising effectors for anticancer therapy. Considering their participation in tumorigenesis, their utilization in drug development has been the focus of both industry and academic research efforts. These efforts have been developing in two main directions; (i) utilizing GAGs as targets of therapeutic strategies and (ii) employing GAGs specificity and excellent physicochemical properties for targeted delivery of cancer therapeutics. This review will comprehensively discuss recent developments and the broad potential of GAG utilization for cancer therapy.


Assuntos
Glicosaminoglicanos/metabolismo , Animais , Protocolos Antineoplásicos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Heparina/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Nanoestruturas/química
16.
Biomolecules ; 11(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917853

RESUMO

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10-6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10-7 M, 2.15 × 10-8 M and 6.4 × 10-10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.


Assuntos
Antitrombina III/metabolismo , Heparina/metabolismo , Antitrombina III/química , Antitrombina III/genética , Sítios de Ligação , Feminino , Heparina/química , Humanos , Imunoeletroforese , Cinética , Masculino , Simulação de Dinâmica Molecular , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ressonância de Plasmônio de Superfície
17.
Angew Chem Int Ed Engl ; 60(29): 15870-15878, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33860605

RESUMO

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.


Assuntos
Antivirais/metabolismo , Heparina/metabolismo , Poliéster Sulfúrico de Pentosana/metabolismo , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Chlorocebus aethiops , Heparina/química , Humanos , Simulação de Dinâmica Molecular , Poliéster Sulfúrico de Pentosana/química , Polímeros/química , Polímeros/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Eletricidade Estática , Células Vero
18.
Methods Mol Biol ; 2263: 183-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877598

RESUMO

Quartz crystal microbalance with dissipation monitoring (QCM-D) is one of the most widely used techniques for the deposition of lipid layers and provides a useful tool for protein-ligand analysis. By using functionalized lipids, for example, with nitrilotriacetic acid (NTA) or biotin, one can couple a molecule to the surface to investigate ligand interactions. Using lipid layers in this way allows for the analysis of complex binding events such as conformational changes, fibrillation, and hierarchical clustering on the surface, which is difficult to interpret with conventional surface sensor techniques. Deposition of lipids and subsequent molecular interactions are easily monitored using both the frequency and the dissipation, which have distinct features in bilayer formation and make QCM-D the ideal technique to use. Here we describe the formation of biotinylated lipid bilayers using two different types of lipids and the subsequent addition of avidin, which can then be used as a basis for linking biotinylated molecules to the surface. These protocols can be adapted to use other lipid moieties and linking chemistries.


Assuntos
Heparina/metabolismo , Bicamadas Lipídicas/metabolismo , Técnicas de Microbalança de Cristal de Quartzo/métodos , Avidina/química , Fenômenos Biofísicos , Técnicas Biossensoriais , Biotina/química , Bicamadas Lipídicas/química , Ligação Proteica , Ressonância de Plasmônio de Superfície
19.
Biochemistry ; 60(15): 1201-1213, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33822598

RESUMO

Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction. To resolve these apparently conflicting findings, we engineered variants in which disulfides designed to block the RCL conformational change were combined with constitutively activating mutations. Our findings demonstrate that while a reversible constitutive activation can be engineered in variants that retain the native RCL-core interaction, engineered disulfides that lock the RCL native conformation can also block heparin allosteric activation. Such findings support a three-state allosteric activation model in which constitutive activating mutations stabilize an intermediate-activated state wherein core conformational changes and a major activation have occurred without the release of the RCL from the core but with a necessary repositioning of the RCL to allow productive engagement with an exosite. Rigid disulfide bonds that lock the RCL native conformation block heparin activation by preventing both RCL repositioning in the intermediate-activated state and the release of the RCL from the core in the fully activated state.


Assuntos
Antitrombinas/química , Antitrombinas/metabolismo , Heparina/metabolismo , Regulação Alostérica , Humanos , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica
20.
Int J Biol Macromol ; 180: 470-483, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33745974

RESUMO

Fibroblast growth factor receptors (FGFRs) are integral membrane proteins involved in various biological processes including proliferation, migration and apoptosis. There are a number of regulatory mechanisms of FGFR signaling, which tightly control the specificity and duration of transmitted signals. The effect of the FGFRs spatial distribution in the plasma membrane on receptor-dependent functions is still largely unknown. We have demonstrated that oligomerization of FGF1 with coiled-coil motifs largely improves FGF1 affinity for FGFRs and heparin. Set of developed FGF1 oligomers evoked prolonged activation of FGFR1 and receptor-downstream signaling pathways, as compared to the wild type FGF1. The majority of obtained oligomeric FGF1 variants showed increased stability, enhanced mitogenic activity and largely improved internalization via FGFR1-dependent endocytosis. Importantly, FGF1 oligomers with the highest oligomeric state exhibited reduced ability to stimulate FGFR-dependent glucose uptake, while at the same time remained hyperactive in the induction of cell proliferation. Our data implicate that oligomerization of FGF1 alters the biological activity of the FGF/GFR1 signaling system. Furthermore, developed FGF1 oligomers, due to improved stability and proliferative potential, can be applied in the regenerative medicine or as drug delivery vehicles in the ADC approach against FGFR1-overproducing cancers.


Assuntos
Proliferação de Células , Fator 1 de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Células 3T3-L1 , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Fator 1 de Crescimento de Fibroblastos/química , Heparina/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Ligação Proteica , Multimerização Proteica
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