RESUMO
KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.
Assuntos
Proteínas de Bactérias/química , Klebsiella pneumoniae/química , Lipoproteínas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.
Assuntos
Bacteroides/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Dobramento de Proteína , Sequência de Aminoácidos , Bacteroides/genética , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
Assuntos
Hemoglobina Fetal/genética , Filtração/métodos , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Ribonucleoproteínas/genética , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Eletroporação/métodos , Feminino , Hemoglobina Fetal/metabolismo , Filtração/economia , Filtração/instrumentação , Terapia Genética/economia , Terapia Genética/instrumentação , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Membranas Artificiais , Camundongos , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Transplante AutólogoRESUMO
Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for â¼1.5% of most prokaryotic genomes and are estimated to be involved in â¼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.