Modulation of mitotic progression and cell cycle checkpoints by phosphorylation-dependent protein-protein interactions

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.Alteration of mitotic gene function has recently been discovered to play a key role in tumor formation and cancer progression through the induction of chromosomal aberrations and genomic instability. Polo-like-kinase-1 is a critical mitotic regulator, overexpressed in human tumors, that functions in mitotic entry after cellular stress, centrosome maturation, mitotic spindle control, and cytokinesis, which are all disregulated in cancer cells. To study the role of Polo-like kinases we took advantage of the recent discovery that the polo-box domain of Polo-like kinases is a phosphorylation-dependent binding module that regulates targeting of Polo-like kinases to their substrates. To identify the interactors of Polo-box domains we developed and performed a mitotic-specific yeast two hybrid and a pulldown mass spectrometry screen. This yielded a large number of specific interactors known to be involved in a vast variety of mitotic processes including those previously described to be involved in tumor progression. We demonstrate that Polo-like kinase regulation of cytokinesis-specific guanine-nucleotide exchange factors for the small G-protein Rho is necessary for proper actomyosin ring contraction and cytokinesis. Additionally we demonstrate that Polo-like-kinase-1 directly regulates the activity of the Rho-effector-kinase ROCK2, and thus Polo-like kinases modulate Rho signaling both upstream and downstream of Rho during cytokinesis. In addition to Polo-box domains we also worked on two other phosphorylation-dependent binding domains involved in cell cycle checkpoints that become disregulated in cancer cells, tandem BRCT domains and WW domains.(cont.) We examined the molecular basis for phosphorylation-dependent recognition by the tandem BRCT domains of BRCA1 through oriented-peptide-library screening and determination of an X-ray crystal structure of the domain bound to a phosphopeptide. This allowed us to rationalize why inherited mutations within the tandem BRCT domains of BRCA1 promote breast and ovarian cancer in humans. Secondly, we assayed WW domains that were generated from in silicon determined sequences for natural-like function to more fully understand the folding and binding requirements of this domain class. All three domains (tandem BRCT domains, Polo-box domains, and WW domains) are attractive targets for cancer therapeutics as they participate in control of processes necessary for genomic stability that become disregulated in cancer.by Drew M. Lowery.Ph.D

The NDR/LATS Family Kinase Cbk1 Directly Controls Transcriptional Asymmetry

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

F62 Validation of digital assessment of physical activity in Huntington’s disease: comparing fitbit charge 4 step count with research-grade accelerometers

Background Physical activity has been implicated in improving symptom management and quality of life in Huntington’s disease (HD). Verifying the role of physical activity in HD requires accurate quantification of exercise metrics. Aim To assess the reliability and validity of Fitbit® Charge 4 step-count compared to research accelerometers and observer count in HD patients. Methods 17 manifest HD participants completed two indoor 2-minute walking tests (2MWT) while wearing a Fitbit® charge 4, and an ActiGraph GTX9 on their non-dominant wrist, ActivPAL4™ on both anterior thighs, and ActiGraph GTX9 on both anterior shanks. Steps were manually counted from video recordings of the 2MWTs. Step-counts for devices were obtained from their proprietary software. Intraclass correlation coefficients (ICC) determined Fitbit and observer reliability, whilst Bland-Altman (B-A) analysis demonstrated monitor agreement. Results Intra-rater reliability of researcher count from repeat trials was excellent (ICC 0.95/n = 17) meanwhile, Fitbit yielded good reliability (ICC 0.81/n = 16). B-A plots displayed greatest agreement between Fitbit and manual count (bias -4.7/SD 36.2). Average monitor count revealed largest consistencies with ActivPAL4 and Fitbit (bias -3.4/SD 37.4) whereas, wrist-worn ActiGraph deviated markedly (bias 4.9/SD 66.9). Motor symptom severity did not influence average Fitbit count although, upper extremity devices occasionally underestimated steps produced. Conclusions Whilst superior step-count accuracy was seen with devices placed on lower limbs, these data show that Fitbit charge 4 may be a reliable device for monitoring steps in manifest HD

F61 Validation of a gait event detection algorithm during overground walking in Huntington's disease

Background Objective evaluation of gait impairment in Huntington’s Disease (HD) is challenging both in clinical trials and clinical practice. Algorithms, such as Teager-Kaiser Gait Event Detection (TKGED), enable detection of initial (IC) and terminal (TC) contact, using accelerometers on the shanks [1]. TKGED has not been validated in HD despite known accuracy in healthy individuals. Aim To assess the performance of TKGED using shank and thigh acceleration signals in participants with HD. Methods Seventeen participants performed two 2-minute walking tests, wearing accelerometers on shanks (ActiGraph GTX9, 100Hz) and thighs (ActivPAL4, 40Hz). Video data were recorded as the criterion measure. To obtain IC and TC points, video data were manually annotated and TKGED was applied to accelerometer signals. Step counts and stride, stance and swing times were estimated for each sensor. Intraclass correlation coefficients (ICC) determined agreement between video and accelerometer step counts. Step count differences were assessed using coefficients of variation (CV) and signed rank tests. Kruskal-Wallis tests assessed differences between video and accelerometer stride, stance and swing times. Results Excellent agreement was observed for step counts between video and both shank (ICC=0.97, CV=4.0%, p=0.21) and thigh (ICC=0.94, CV=5.3%, p=0.23) accelerometers (Figure 1). Compared with video annotation, TKGED tended to underestimate stance time and overestimate swing time (p<0.001), but yielded accurate estimates of stride time (p=0.28)