RESUMO
Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.
Assuntos
Amiloide/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Doença de Parkinson/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Tomografia com Microscopia Eletrônica , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Técnicas In Vitro , Cinética , Chaperonas Moleculares/metabolismo , Doença de Parkinson/etiologia , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Solubilidade , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody-peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells' proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide-thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol-maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody-drug conjugates.
Assuntos
Antineoplásicos , Imunoconjugados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisteína/química , Imunoconjugados/química , Imunoconjugados/farmacologia , Maleimidas/química , Polietilenoglicóis , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Compostos de SulfidrilaRESUMO
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Agregados Proteicos , Animais , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP70/química , Humanos , Modelos Moleculares , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/prevenção & controle , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.
Assuntos
Alfa-Amanitina/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Alfa-Amanitina/química , Animais , Linhagem Celular Tumoral , Endocitose , Fator 2 de Crescimento de Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Oligopeptídeos/química , Estrutura Secundária de Proteína , Transdução de SinaisRESUMO
Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy.
Assuntos
Fator 1 de Crescimento de Fibroblastos/antagonistas & inibidores , Biblioteca de Peptídeos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Clonais , Clonagem Molecular , Reações Cruzadas , Escherichia coli/genética , Escherichia coli/metabolismo , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Camundongos , Células NIH 3T3 , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genéticaRESUMO
Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody-drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are over-expressed in several types of lung, breast, and gastric cancers and are therefore an attractive molecular target. Recently, we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield and led to nonhomogeneous products. To improve the conjugation, we introduced a novel Lys-Cys-Lys motif at either FGF1 terminus, which increased cysteine reactivity and allowed us to obtain an FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines, we confirmed specific cytotoxity of the obtained C-terminal FGF1-vcMMAE conjugate and its selective endocytososis as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively charged amino acids could be used universally to improve the efficiency of the site-specific chemical modification of other proteins.
Assuntos
Antineoplásicos/química , Sistemas de Liberação de Medicamentos/métodos , Fator 1 de Crescimento de Fibroblastos/química , Oligopeptídeos/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisteína/química , Endocitose/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP.AlF(4)(-)-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/química , Compostos de Alumínio/metabolismo , Cristalografia por Raios X , Fluoretos/química , Fluoretos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Mathanococcus , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Canais de Translocação SEC , Relação Estrutura-AtividadeRESUMO
Targeted therapies are a promising alternative to conventional chemotherapy, with an increasing number of therapeutics targeting specific molecular aberrancies in cancer cells. One of the emerging targets for directed cancer treatments is fibroblast growth factor receptors (FGFRs), which are known to be involved in the pathogenesis and progression of multiple cancer types, specially in lung, bladder, and breast cancers. Here, we are demonstrating the development of the FGFR1-targeting agent based on the interactome screening approach, based on the isolation of binding regions from ligands interacting with the receptor. The parallel analysis by FGFR1 pull-down of chymotryptic peptides coupled with MS analysis, and PepSpot analysis yielded equivalent peptide sequences from FGF4, one of the FGFR1 ligands. Three sequences served as a basis for peptibody (Fc-fusion) generation, to overcome clinical limitations of peptidic agents, and two of them showed favorable FGFR1-binding in vitro and FGFR1-dependent internalization into cells. To validate if developed FGFR1-targeting peptibodies can be used for drug delivery, similar to the well-established concept of antibody-drug conjugates (ADCs), peptibodyF4_1 was successfully conjugated with monomethylauristatin E (MMAE), and has shown significant and specific toxicity toward FGFR1-expressing lung cancer cell lines, with nanomolar EC50 values. Essentially, the development of new effective FGFR1 binders that comprise the naturally occurring FGFR-recognition peptides and Fc region ensuring high plasma stability, and long bloodstream circulation is an interesting strategy expanding targeted anticancer agents' portfolio. Furthermore, identifying peptides effectively binding the receptor from sequences of its ligands is not limited to FGFRs and is an approach versatile enough to be a basis for a new peptide/peptibodies development strategy.
RESUMO
Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Maleimidas/química , Compostos de Sulfidrila/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Domínios Proteicos , Engenharia de ProteínasRESUMO
Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1.fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1.FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Heparina/química , Receptores de Fatores de Crescimento de Fibroblastos/química , Animais , Sítios de Ligação , Proliferação de Células , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Transdução de Sinais , TermodinâmicaRESUMO
Comparison of the crystal structures of two pentadehydropeptides containing DeltaPhe residues, namely (Z,Z)-N-(tert-butoxycarbonyl)glycyl-alpha,beta-phenylalanylglycyl-alpha,beta-phenylalanylglycine (or Boc(0)-Gly(1)-Delta(Z)Phe(2)-Gly(3)-Delta(Z)Phe(4)-Gly(5)-OH) methanol solvate, C(29)H(33)N(5)O(8) x CH(4)O, (I), and (E,E)-N-(tert-butoxycarbonyl)glycyl-alpha,beta-phenylalanylglycyl-alpha,beta-phenylalanylglycine (or Boc(0)-Gly(1)-Delta(E)Phe(2)-Gly(3)-Delta(E)Phe(4)-Gly(5)-OH), C(29)H(33)N(5)O(8), (II), indicates that the Delta(Z)Phe residue is a more effective inducer of folded structures than the Delta(E)Phe residue. The values of the torsion angles phi and psi show the presence of two type-III' beta-turns at the Delta(Z)Phe residues and one type-II beta-turn at the Delta(E)Phe residue. All amino acids are linked trans to each other in both peptides. Beta-turns present in the peptides are stabilized by intramolecular 4-->1 hydrogen bonds. Molecules in both structures form two-dimensional hydrogen-bond networks parallel to the (100) plane.
Assuntos
Oligopeptídeos/química , Fenilalanina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody-drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody-drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.
RESUMO
Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Mutação , Proteínas Recombinantes/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Meia-Vida , Humanos , Ligação de Hidrogênio , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Overexpression of fibroblast growth factor receptor 1 (FGFR1) is a common aberration in lung and breast cancers and has necessitated the design of drugs targeting FGFR1-dependent downstream signaling and FGFR1 ligand binding. To date, the major group of drugs being developed for treatment of FGFR1-dependent cancers are small-molecule tyrosine kinase inhibitors; however, the limited specificity of these drugs has led to increasing attempts to design molecules targeting the extracellular domain of FGFR1. Here, we used the phage display technique to select cyclic peptides F8 (ACSLNHTVNC) and G10 (ACSAKTTSAC) as binders of the fibroblast growth factor 1 (FGF1)-FGFR1 interface. ELISA and in vitro cell assays were performed to reveal that cyclic peptide F8 is more effective in preventing the FGF1-FGFR1 interaction, and also decreases FGF1-induced proliferation of BA/F3 FGFR1c cells by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Therefore, cyclic peptide F8 can act as a FGF1-FGFR1 interaction antagonist, and may be suitable for further development for potential use in therapies against FGFR1-expressing cancer cells.
Assuntos
Bacteriófagos/metabolismo , Fator 1 de Crescimento de Fibroblastos/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos Cíclicos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Linhagem Celular , Técnicas de Visualização da Superfície Celular , Perfilação da Expressão Gênica , Camundongos , Células NIH 3T3 , Transdução de SinaisRESUMO
Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin αvß3 through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin αvß3. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Integrina alfaVbeta3/metabolismo , Estabilidade Proteica , Proteólise , Animais , Sítios de Ligação , Fator 1 de Crescimento de Fibroblastos/genética , Heparina/química , Humanos , Integrina alfaVbeta3/química , Cinética , Camundongos , Mutação , Células NIH 3T3 , Ligação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/químicaRESUMO
Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified in vitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J-protein co-chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J-protein of Caenorhabditis elegans work together to resolve protein aggregates both in vivo and in vitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J-proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat-induced protein aggregates and polyQ aggregates that form in an age-dependent manner. Cooperation between class A and B J-proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J-proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70-based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.
Assuntos
Proteínas de Choque Térmico/metabolismo , Agregados Proteicos/fisiologia , Envelhecimento , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Estresse FisiológicoRESUMO
Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.
Assuntos
Proteínas de Escherichia coli/química , Evolução Molecular , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Agregados Proteicos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Filogenia , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody-drug conjugates. The FGF1V-valine-citrulline-MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V-vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality.
Assuntos
Antineoplásicos/química , Desenho de Fármacos , Fator 1 de Crescimento de Fibroblastos/química , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/uso terapêutico , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are members of the Hsp100 family of ring-forming hexameric AAA+ chaperones that promote the solubilization of aggregated proteins and the propagation of prions. ClpB and Hsp104 cooperate with cognate Hsp70 chaperones for substrate targeting and activation of ATPase and substrate threading, achieved by transient Hsp70 binding to the repressing ClpB/Hsp104 M-domain. Fundamental differences in ATPase regulation and disaggregation mechanisms have been reported; however, these differences are raising doubts regarding the working principle of this AAA+ chaperone. In particular, unique functional plasticity was suggested to specifically enable Hsp104 to circumvent Hsp70 requirement for derepression in protein disaggregation and prion propagation. We show here that both ClpB and Hsp104 cooperation with Hsp70 is crucial for efficient protein disaggregation and, in contrast to earlier claims, cannot be circumvented by activating M-domain mutations. Activation of ClpB and Hsp104 requires two signals, relief of M-domain repression and substrate binding, leading to increased ATPase subunit coupling. These data demonstrate that ClpB and Hsp104 operate by the same basic mechanism, underscore a dominant function of Hsp70 in regulating ClpB/Hsp104 activity, and explain a plethora of in vivo studies showing a crucial function of Hsp70 in proteostasis and prion propagation.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Endopeptidase Clp , Agregados Proteicos , Mapeamento de Interação de ProteínasRESUMO
Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy.