19 research outputs found
Irreversible chemical steps control intersubunit dynamics during translation
The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation
Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca2+-binding proteins in Dictyostelium discoideum
Mass Spectrometric Characterization of Transglutaminase Based Site-Specific Antibody–Drug Conjugates
Antibody drug conjugates (ADCs) are
becoming an important new class
of therapeutic agents for the treatment of cancer. ADCs are produced
through the linkage of a cytotoxic small molecule (drug) to monoclonal
antibodies that target tumor cells. Traditionally, most ADCs rely
on chemical conjugation methods that yield heterogeneous mixtures
of varying number of drugs attached at different positions. The potential
benefits of site-specific drug conjugation in terms of stability,
manufacturing, and improved therapeutic index has recently led to
the development of several new site-specific conjugation technologies.
However, detailed characterization of the degree of site specificity
is currently lacking. In this study we utilize mass spectrometry to
characterize the extent of site-specificity of an enzyme-based site-specific
antibody–drug conjugation technology that we recently developed.
We found that, in addition to conjugation of the engineered site,
a small amount of aglycosylated antibody present in starting material
led to conjugation at position Q295, resulting in approximately 1.3%
of off-target conjugation. Based on our detection limits, we show
that Q295N mutant eliminates the off-target conjugation yielding highly
homogeneous conjugates that are better than 99.8% site-specific. Our
study demonstrates the importance of detailed characterization of
ADCs and describes methods that can be utilized to characterize not
only our enzyme based conjugates, but also ADCs generated by other
conjugation technologies
Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
<div><p>The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency <i>in vitro</i> and <i>in vivo</i>. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.</p></div
Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
Degradation of the C-terminal portion of the PEG6-C2-MMAD payload in the plasma of different species.
<p>Degradation is calculated as percentage of payload cleaved. Calculations are based on DAR values obtained from HIC analysis of the Site A-PEG6-2-MMAD conjugate before and after incubation in plasma for 4.5 days in three independent experiments.</p