Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis.

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.

A Tunable Brake for HECT Ubiquitin Ligases.

The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

Ubiquitin Ligase Activities of WWP1 Germline Variants K740N and N745S.

WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) N. Engl. J. Med.]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and in vitro enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.

Impact of COVID-19 Restrictions on Demographics and Outcomes of Patients Undergoing Medically Necessary Non-Emergent Surgeries During the Pandemic.

BACKGROUND: The COVID-19 pandemic has resulted in large-scale healthcare restrictions to control viral spread, reducing operating room censuses to include only medically necessary surgeries. The impact of restrictions on which patients undergo surgical procedures and their perioperative outcomes is less understood. METHODS: Adult patients who underwent medically necessary surgical procedures at our institution during a restricted operative period due to the COVID-19 pandemic (March 23-April 24, 2020) were compared to patients undergoing procedures during a similar time period in the pre-COVID-19 era (March 25-April 26, 2019). Cardinal matching and differences in means were utilized to analyze perioperative outcomes. RESULTS: 857 patients had surgery in 2019 (pre-COVID-19) and 212 patients had surgery in 2020 (COVID-19). The COVID-19 era cohort had a higher proportion of patients who were male (61.3% vs. 44.5%, P < 0.0001), were White (83.5% vs. 68.7%, P < 0.001), had private insurance (62.7% vs. 54.3%, p 0.05), were ASA classification 4 (10.9% vs. 3%, P < 0.0001), and underwent oncologic procedures (69.3% vs. 42.7%, P < 0.0001). Following 1:1 cardinal matching, COVID-19 era patients (N = 157) had a decreased likelihood of discharge to a nursing facility (risk difference-8.3, P < 0.0001) and shorter median length of stay (risk difference-0.6, p 0.04) compared to pre-COVID-19 era patients. There was no difference between the two patient cohorts in overall morbidity and 30-day readmission. CONCLUSIONS: COVID-19 restrictions on surgical operations were associated with a change in the racial and insurance demographics in patients undergoing medically necessary surgical procedures but were not associated with worse postoperative morbidity. Further study is necessary to better identify the causes for patient demographic differences.

Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation.

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN antibody, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN antibody.

Enzyme-catalyzed expressed protein ligation.

Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms.

N-Hydroxysuccinimide (NHS)-esters are widely used to label proteins nonselectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transform NHS-esters into site-specific protein labeling on N-terminal Cys residues. MESNA addition converts NHS-esters to chemoselective thioesters for N-Cys modification. This labeling strategy was applied to clarify mechanistic features of the ubiquitin E3 ligase WWP2 including its interaction with one of its substrates, the tumor suppressor PTEN, as well as its autoubiquitination molecularity. We propose that this convenient protein labeling strategy will allow for an expanded application of NHS-esters in biochemical investigation.

Surgical Management of Early-Stage Esophageal Adenocarcinoma Based on Lymph Node Metastasis Risk.

BACKGROUND: In early-stage esophageal adenocarcinoma (EAC), esophagectomy improves staging but also increases mortality compared with endoscopic resection. Our objective was to quantify esophagectomy mortality and lymph node metastasis (LNM) risk in early-stage EAC to improve surgical treatment allocation. METHODS: We identified National Cancer Database (2004-2014) patients with nonmetastatic, Tis, T1a, or T1b EAC who had primary surgical resection and microscopic examination of at least 15 lymph nodes. Univariate and multivariable logistic regression identified predictors of LNM. Cox regression identified predictors of death. The Kaplan-Meier method predicted overall survival (OS). RESULTS: In 782 patients, LNM rates were: all patients 13.8%, Tis 0%, T1a 3.6%, T1b 23.4%. Independent predictors of LNM were submucosal invasion, lymphovascular invasion (LVI), decreasing differentiation, and tumor size ≥ 2 cm (P < 0.05). For T1a tumors with poor differentiation or size ≥ 2 cm, LNM rates were 10.2 and 6.7%, respectively; 90-day mortality was 3.1%. The LNM rate in well differentiated T1b tumors < 2 cm was 4.2%; 90-day mortality was 6.0%. Estimated 5-year OS was 80.2% versus 64.4% (T1a vs. T1b). LNM increased risk of death for T1a (hazard ratio [HR] 8.52, 95% confidence interval [CI] 3.13-23.22, P < 0.001) and T1b tumors (HR 2.52, 95% CI 1.59-4.00, P < 0.001). CONCLUSIONS: In T1a EAC with poor differentiation or size ≥ 2 cm, esophagectomy should be considered, whereas in T1b EAC with low-risk features (well-differentiated T1b EAC < 2 cm without LVI), endoscopic resection may be sufficient. Treatment guidelines for early-stage EAC should include all high-risk tumor features for LNM and stage-specific esophagectomy mortality.

Implementation of a systematic culturing program to monitor the efficacy of endoscope reprocessing: outcomes and costs.

BACKGROUND AND AIMS: In 2015, the U.S. Food and Drug Administration and Centers for Disease Control and Prevention (CDC) issued guidance for duodenoscope culturing and reprocessing in response to outbreaks of carbapenem-resistant Enterobacteriaceae (CRE) duodenoscope-related infections. Based on this guidance, we implemented best practices for reprocessing and developed a systematic process for culturing endoscopes with elevator levers. The aim of this study is to report the outcomes and direct costs of this program. METHODS: First, clinical microbiology data from 2011 to 2014 were reviewed retrospectively to assess for possible elevator lever-equipped endoscope-related CRE infections. Second, a program to systematically culture elevator lever-equipped endoscopes was implemented. Each week, about 25% of the inventory of elevator lever-equipped endoscopes is cultured based on the CDC guidelines. If any cultures return bacterial growth, the endoscope is quarantined pending repeat culturing. The costs of the program, including staff time and supplies, have been calculated. RESULTS: From 2011 to 2014, none of 17 patients with documented CRE infection had undergone ERCP or endoscopic ultrasound in the previous 36 months. From June 2015 to September 2016, 285 cultures were performed. Three (1.1%) had bacterial growth, 2 with skin contaminants and 1 with an oral contaminant. The associated endoscopes were quarantined and reprocessed, and repeat cultures were negative. The total estimated cost of our program for an inventory of 20 elevator lever-equipped endoscopes was $30,429.60 per year ($1521.48 per endoscope). CONCLUSIONS: This 16-month evaluation of a systematic endoscope culturing program identified a low rate of positive cultures after elevator lever endoscope reprocessing. All positive cultures were with non-enteric microorganisms. The program was of modest cost and identified reprocessing procedures that may have led to a low rate of positive cultures.