SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA ...binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery.
SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.
Abstract
Background: Breast cancer remains the most frequently diagnosed cancer among women worldwide, accounting for a quarter of all diagnoses. Despite advances in treatment and symptom management, ...the majority of women will experience some form of drug-related toxicity, psychosocial distress, and subsequent impairments in their quality of life (QoL). Distress and impairments in QoL can interfere with treatment adherence, while engagement in health promoting behaviors and effective management of symptoms has been associated with improved QoL, adherence and increased survival. The utilization of QoL or other Patient Reported Outcome (PRO) measures in clinical trials remains inconsistent, and no uniformly accepted measure exists to integrate QoL data into the assessment of therapeutic agents. There are two goals of the I-SPY 2 QoL Pilot Study: 1) To demonstrate the reporting of an integrated utility-based QOL score, the PROPr, within a novel longitudinal approach that provides a single numerical index of QoL; 2) Generate a Clinical Benefit Index (CBI), a single composite score that integrates the longitudinal PROPr score with a clinical efficacy score, RCB index, to provide a measure that could go beyond clinical efficacy in the evaluation of therapeutic agents in the I-SPY 2 TRIAL. Methods: Study participants were part of the I-SPY 2 TRIAL assessing novel neoadjuvant therapies added to standard chemotherapy in the treatment of Stage 2/3 breast cancer. Participants completed a validated QoL measure at three time points: baseline, prior to surgery, and 1-month post-surgery. QoL was assessed using the NIH PROMIS measure (physical function (four items), anxiety (eight items), depression (eight items), fatigue (eight items), applied cognition (eight items) and social roles (four items)) and results at each time point used to calculate the PROPr, a single utility-based index score to assess overall quality of life. PROPr index utility scores were used to generate a single longitudinal QoL score based on area under the curve modeling. Clinical efficacy was assessed based on the residual cancer burden (RCB) observed at the time of surgery. The CBI was generated by plotting RCB index against the longitudinal PROPr index for each participant and study arm. Results: Only a fraction (n=107) of all patients had complete data across study timepoints and were included in our analyses, and thus our data represent a proof of concept. Patients on the control arm were treated with Paclitaxel followed by anthracycline (AC). Patients in the pilot were assigned either the control arm or six experimental drug arms. The longitudinal PROPr utility index demonstrated a range of outcomes, with some arms more challenging to tolerate, and others much better, ranging from 0.67 to 1.16. The RCB index of the seven study arms ranged from 0.49 to 1.99. The CBI, an integration of the longitudinal PROPr and RCB indexes, also demonstrated a range from 0.43 to 1.60. Conclusion: We are reporting the development of a novel, valid and standardized QoL assessment that should be a routine part of clinical trials in oncology. This proof of concept study suggests that that calculation of the CBI is feasible and can reveal differences in the clinical profiles of therapeutic agents, both in terms of QoL and overall integration of clinical efficacy and QoL. The CBI represents a novel approach to providing summary data that can be easily interpreted as part of clinical trial outcome data. Ideally, these integrated assessments would provide a more comprehensive evaluation of investigational therapies, and ultimately help inform treatment decision discussions between patients and providers. Moving forward, electronic PRO data will be collected as part of routine care in the I-SPY 2 TRIAL, thus enabling the longitudinal PROPr and CBI scores to be generated for every agent evaluated.
Citation Format: Amrita Basu, Errol J. Philip, Barry Dewitt, Janel Hanmer, Aheli Chattopadhyay, Christina Yau, Smita Asare, Karyn Digiorgio, Ruby Singhrao, Adam Asare, Jane Perlmutter, I-SPY 2 Consortium, Michelle Melisko, Laura Esserman. The clinical benefit index: A pilot study integrating treatment efficacy and quality of life in oncology clinical trials abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-12-06.
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