RESUMO
Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters "CPPs" (cholesterol concentration, total flow rate "TFR" and flow rate ratio "FRR"), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design).
Assuntos
Colesterol , Lipossomos , Microfluídica , Polietilenoglicóis , Colesterol/química , Polietilenoglicóis/química , Microfluídica/métodos , Doxorrubicina/química , Doxorrubicina/análogos & derivados , Fosfatidiletanolaminas/química , Tamanho da Partícula , Química Farmacêutica/métodos , Esfingomielinas/química , Tecnologia Farmacêutica/métodos , Composição de Medicamentos/métodos , Fosfatidilcolinas/química , Sistemas de Liberação de MedicamentosRESUMO
EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1.
Assuntos
Glicoproteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Amidas/química , Complemento C1q/química , Complemento C1q/genética , Simulação por Computador , Cristalografia por Raios X , Humanos , Glicoproteínas de Membrana/genética , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.
Assuntos
Movimento Celular/fisiologia , Integrina alfa4beta1/química , Integrina alfa4beta1/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Pressão Sanguínea/fisiologia , Adesão Celular/fisiologia , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Homeostase/fisiologia , Humanos , Integrina alfa4beta1/genética , Células Jurkat , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Relação Estrutura-Atividade , Trofoblastos/metabolismo , Útero/metabolismoRESUMO
Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/isolamento & purificação , Genes Sintéticos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Cromatografia Líquida , Dicroísmo Circular , Proteínas de Drosophila , Escherichia coli/genética , Expressão Gênica/genética , Genes Sintéticos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/química , Proteínas de Membrana/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Receptores Notch/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Serrate-JaggedRESUMO
The solution structure of human beta(2)-microglobulin (beta(2)-m) was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand C' and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of beta(2)-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained.
Assuntos
Amiloide/química , Conformação Proteica , Microglobulina beta-2/química , Cristalografia por Raios X , Estabilidade de Medicamentos , Evolução Molecular , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Soluções , Termodinâmica , Microglobulina beta-2/genéticaRESUMO
Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.
Assuntos
Microglobulina beta-2/química , Amiloide/metabolismo , Amiloidose/metabolismo , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Desnaturação Proteica , Relação Estrutura-Atividade , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
The solution structure of human beta2-microglobulin (beta2-m), the nonpolymorphic component of class I major histocompatibility complex (MHC-I), was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to previous structural data obtained from the NMR secondary structure of the isolated protein and the crystal structure of MHC-I, in which the protein is associated to the heavy-chain component, several differences are observed. The most important rearrangements were observed for (1) strands V and VI (loss of the C-terminal and N-terminal end, respectively), (2) interstrand loop V-VI, and (3) strand I, including the N-terminal segment (displacement outward of the molecular core). These modifications can be considered as the prodromes of the amyloid transition. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches at the interface with heavy chain and the nearby region at the surface charge cluster of the C-terminal segment. As a result, the molecule is placed in a state in which even minor charge and solvation changes in response to pH or ionic-strength variations can easily compromise the hydrophobic/hydrophilic balance and trigger the transition into a partially unfolded intermediate that starts with unpairing of strand I and leads to polymerization and precipitation into fibrils or amorphous aggregates. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding, which is shown to occur primarily at His 31 and involve partially also His 13, the next available His residue along the partial unfolding pathway.