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1.
Elife ; 92020 08 27.
Article in English | MEDLINE | ID: mdl-32851975

ABSTRACT

Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.


Subject(s)
Neurons , Nuclear Receptor Subfamily 1, Group F, Member 2 , Somatosensory Cortex , Animals , Antigens, Surface/chemistry , Antigens, Surface/genetics , Antigens, Surface/metabolism , Female , Male , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Neurons/chemistry , Neurons/cytology , Neurons/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 2/chemistry , Nuclear Receptor Subfamily 1, Group F, Member 2/genetics , Nuclear Receptor Subfamily 1, Group F, Member 2/metabolism , Somatosensory Cortex/chemistry , Somatosensory Cortex/cytology , Somatosensory Cortex/metabolism , Somatosensory Cortex/physiology , Thalamus/chemistry , Thalamus/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/genetics
2.
Biochemistry ; 55(23): 3315-28, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27194363

ABSTRACT

A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive.


Subject(s)
Algorithms , Aspartic Acid/chemistry , Glutamic Acid/chemistry , Proteins/chemistry , Humans , Hydrogen Bonding , Hydrolysis , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Thermodynamics
3.
MAbs ; 8(1): 99-112, 2016.
Article in English | MEDLINE | ID: mdl-26514585

ABSTRACT

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.


Subject(s)
Bevacizumab/chemistry , Bevacizumab/genetics , Protein Aggregates , Protein Engineering/methods , Amino Acid Motifs , Bevacizumab/biosynthesis , Cell Line , Humans
4.
Mol Pharm ; 12(5): 1443-55, 2015 May 04.
Article in English | MEDLINE | ID: mdl-25871775

ABSTRACT

Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2-8 and 0-300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions.


Subject(s)
Antibodies, Monoclonal/chemistry , Colloids/chemistry , Immunoglobulin G/chemistry , Chromatography, Gel , Circular Dichroism , Dynamic Light Scattering , Humans , Hydrogen-Ion Concentration , Protein Binding , Protein Conformation , Spectrometry, Fluorescence
5.
Plant Physiol ; 160(1): 118-34, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22732244

ABSTRACT

Members of the Bric-a-Brac/Tramtrack/Broad Complex (BTB) family direct the selective ubiquitylation of proteins following their assembly into Cullin3-based ubiquitin ligases. Here, we describe a subfamily of nucleus-localized BTB proteins encoded by the LIGHT-RESPONSE BTB1 (LRB1) and LRB2 loci in Arabidopsis (Arabidopsis thaliana) that strongly influences photomorphogenesis. Whereas single lrb1 and lrb2 mutants are relatively normal phenotypically, double mutants are markedly hypersensitive to red light, but not to far-red or blue light, and are compromised in multiple photomorphogenic processes, including seed germination, cotyledon opening and expansion, chlorophyll accumulation, shade avoidance, and flowering time. This red light hypersensitivity can be overcome by eliminating phytochrome B (phyB) and phyD, indicating that LRB1/2 act downstream of these two photoreceptor isoforms. Levels of phyB/D proteins but not their messenger RNAs are abnormally high in light-grown lrb1 lrb2 plants, implying that their light-dependent turnover is substantially dampened. Whereas other red light-hypersensitive mutants accumulate phyA protein similar to or higher than the wild type in light, the lrb1 lrb2 mutants accumulate less, suggesting that LRB1/2 also positively regulate phyA levels in a phyB/D-dependent manner. Together, these data show that the BTB ubiquitin ligases assembled with LRB1/2 function redundantly as negative regulators of photomorphogenesis, possibly by influencing the turnover of phyB/D.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/radiation effects , Light , Nuclear Proteins/metabolism , Phytochrome B/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Apoproteins/genetics , Apoproteins/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Color , Cotyledon/genetics , Cotyledon/physiology , Cotyledon/radiation effects , Epistasis, Genetic , Flowers/physiology , Gene Expression Regulation, Plant , Genetic Loci , Germination , Nuclear Proteins/genetics , Phytochrome/genetics , Phytochrome/metabolism , Phytochrome B/genetics , Plant Development/radiation effects , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Plants, Genetically Modified/radiation effects , Protein Stability , Seeds/genetics , Seeds/physiology , Seeds/radiation effects , Ubiquitin-Protein Ligases/genetics , Ubiquitination
6.
J Pharm Sci ; 101(1): 102-15, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21935950

ABSTRACT

Determining the aggregation propensity of protein-based biotherapeutics is an important step in the drug development process. Typically, a great deal of data collected over a large period of time is needed to estimate the aggregation propensity of biotherapeutics. Thus, candidates cannot be screened early on for aggregation propensity, but early screening is desirable to help streamline drug development. Here, we present a simple molecular computational method to predict the aggregation propensity via hydrophobic interactions, thought to be the most common mechanism of aggregation, and electrostatic interactions. This method uses a new quantity termed Developability Index. It is a function of an antibody's net charge, calculated on the full-length antibody structure, and the spatial aggregation propensity, calculated on the complementarity-determining region structure. Its accuracy is due to the molecular level details and the incorporation of the tertiary structure of the antibody. It is particularly applicable to antibodies or other proteins for which structures are available or could be determined accurately using homology modeling. Applications include the selection of molecules in the discovery or early development process, selection of mutants for stability, and estimation of resources needed for development of a given biomolecule.


Subject(s)
Antibodies/chemistry , Complementarity Determining Regions/chemistry , Computers, Molecular , Drug Discovery/methods , Hydrophobic and Hydrophilic Interactions , Immunoglobulin G/chemistry , Molecular Dynamics Simulation , Protein Structure, Tertiary , Static Electricity
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