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1.
MAbs ; 12(1): 1717265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31980006

RESUMO

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.


Assuntos
Anticorpos Monoclonais , Técnicas de Visualização da Superfície Celular/métodos , Cadeias alfa de Integrinas/antagonistas & inibidores , Integrina beta1 , Animais , Humanos , Camundongos , Biblioteca de Peptídeos
2.
MAbs ; 10(6): 890-900, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110240

RESUMO

Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.


Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão/métodos , Integrinas/química , Modelos Moleculares , Domínios Proteicos , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Desenho Assistido por Computador , Humanos , Interações Hidrofóbicas e Hidrofílicas , Integrinas/metabolismo , Ligação Proteica , Estudos Retrospectivos
3.
Thyroid ; 25(11): 1224-34, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26131603

RESUMO

BACKGROUND: It is of critical clinical importance to select accurately for surgery thyroid nodules at risk for malignancy and avoid surgery on those that are benign. Using alterations in subcellular localization for seven putative biomarker proteins (identified by proteomics), this study aimed to define a specific combination of proteins in surgical tissues that could distinguish benign from malignant nodules to assist in future surgical selection by fine-needle aspiration biopsy (FNAB). METHODS: Immunohistochemical subcellular localization (IHC) analyses of seven proteins were retrospectively performed on surgical tissues (115 benign nodules and 114 papillary-based thyroid carcinomas [TC]), and a risk model biomarker panel was developed and validated. The biomarker panel efficacy was verified in 50 FNAB formalin-fixed and paraffin-embedded cell blocks, and 26 cytosmears were prepared from fresh surgically resected thyroid nodules. RESULTS: Selection modeling using these proteins resulted in nuclear phosphoglycerate kinase 1 (PGK1) loss and nuclear Galectin-3 overexpression as the best combination for distinguishing TC from benign nodules (area under the curve [AUC] 0.96 and 0.95 in test and validation sets, respectively). A computed malignancy score also accurately identified TC in benign and indeterminate nodules (test and validation sets: AUC 0.94, 0.90; specificity 98%, 99%). Its efficacy was confirmed in surgical FNAB cell blocks and cytosmears. CONCLUSION: Using surgical tissues, it was observed that a combination of PGK1 and Galectin-3 had high efficiency for distinguishing benign from malignant thyroid nodules and could improve surgical selection for TC among indeterminate nodules. Further validation in prospective preoperative FNAB will be required to confirm such a clinical application.


Assuntos
Carcinoma Papilar/diagnóstico , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia por Agulha Fina , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Frações Subcelulares/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/patologia , Adulto Jovem
4.
RNA ; 21(6): 1203-16, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25904134

RESUMO

YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ribossômicas/metabolismo , Arginina/metabolismo , Sítios de Ligação , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/química , Guanosina Trifosfato/metabolismo , Lisina/metabolismo , Modelos Moleculares , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Dedos de Zinco
5.
J Mol Biol ; 427(10): 1875-86, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25636407

RESUMO

Rvb1 and Rvb2 are conserved AAA+ (ATPases associated with diverse cellular activities) proteins found at the core of large multicomponent complexes that play key roles in chromatin remodeling, integrity of the telomeres, ribonucleoprotein complex biogenesis and other essential cellular processes. These proteins contain an AAA+ domain for ATP binding and hydrolysis and an insertion domain proposed to bind DNA/RNA. Yeast Rvb1 and Rvb2 proteins oligomerize primarily as heterohexameric rings. The six AAA+ core domains form the body of the ring and the insertion domains protrude from one face of the ring. Conversely, human Rvbs form a mixture of hexamers and dodecamers made of two stacked hexamers interacting through the insertion domains. Human dodecamers adopt either a contracted or a stretched conformation. Here, we found that yeast Rvb1/Rvb2 complexes when assembled in vivo mainly form hexamers but they also assemble as dodecamers with a frequency lower than 10%. Yeast dodecamers adopt not only the stretched and contracted structures that have been described for human Rvb1/Rvb2 dodecamers but also intermediate conformations in between these two extreme states. The orientation of the insertion domains of Rvb1 and Rvb2 proteins in these conformers changes as the dodecamer transitions from the stretched structure to a more contracted structure. Finally, we observed that for the yeast proteins, oligomerization as a dodecamer inhibits the ATPase activity of the Rvb1/Rvb2 complex. These results indicate that although human and yeast Rvb1 and Rvb2 proteins share high degree of homology, there are significant differences in their oligomeric behavior and dynamics.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/química , DNA Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , Cristalografia por Raios X , DNA Helicases/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
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