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Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752


Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
Sci Rep ; 9(1): 13615, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541188


Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.

Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/ultraestrutura , Domínio Catalítico , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/metabolismo , Fenilcetonúrias/genética , Fenilcetonúrias/fisiopatologia , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Raios X
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3277-3285, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28962896


OBJECTIVE: The transcription factor Krüppel-like factor 14 (KLF14) has been associated with type 2 diabetes and high-density lipoprotein-cholesterol (HDL-C) through genome-wide association studies. The mechanistic underpinnings of KLF14's control of metabolic processes remain largely unknown. We studied the physiological roles of KLF14 in a knockout (KO) mouse model. METHODS: Male whole body Klf14 KO mice were fed a chow or high fat diet (HFD) and diet induced phenotypes were analyzed. Additionally, tissue-specific expression of Klf14 was determined using RT-PCR, RNA sequencing, immunoblotting and whole mount lacZ staining. Finally, the consequences of KLF14 loss-of-function were studied using RNA sequencing in tissues with relatively high Klf14 expression levels. RESULTS: KLF14 loss-of-function did not affect HFD-induced weight gain or insulin resistance. Fasting plasma concentrations of glucose, insulin, cholesterol, HDL-C and ApoA-I were also comparable between Klf14+/+ and Klf14-/- mice on chow and HFD. We found that in mice expression of Klf14 was the highest in the anterior pituitary (adenohypophysis), lower but detectable in white adipose tissue and undetectable in liver. Loss of KLF14 function impacted on the pituitary transcriptome with extracellular matrix organization as the primary affected pathway and a predicted link to glucocorticoid receptor signaling. CONCLUSIONS: Whole body loss of KLF14 function in male mice does not result in metabolic abnormalities as assessed under chow and HFD conditions. Mostly likely there is redundancy for the role of KLF14 in the mouse and a diverging function in humans.

Fatores de Transcrição Kruppel-Like/deficiência , Síndrome Metabólica/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Síndrome Metabólica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucocorticoides/metabolismo , Análise de Sequência de RNA