Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

Gender, racial, and socioeconomic disparity of preoperative optimization goals in ventral hernia repair.

BACKGROUND: Preoperative optimization cut-offs are frequently utilized to determine eligibility for elective ventral hernia repair. Our objective was to assess the relationship between gender, race, and socioeconomic status and preoperative optimization goals. METHODS: We queried our institutional database for adults with ventral hernia diagnoses between 2016 and 2021. Demographics, comorbidities, laboratory, and operative data were collected and analyzed. The following cut-offs were used to determine eligibility for elective repair: body mass index (BMI) < 40 kg/m2, no active smoking, and glycated hemoglobin (HbA1c) < 8%. Socioeconomic status was assessed using the Distressed Communities Index. RESULTS: A total of 5638 patients were included [Whites = 4321 (77%), Blacks = 794 (14%), Hispanics = 318 (6%), and other/unknown 205 (4%)]. Median age was 61 years and 50% were male. Most common hernia types were umbilical (36%) and incisional (20%). 10% had BMI > 40 kg/m2, 9% were active smokers and 4% had HbA1c > 8%. 21% of all patients did not meet the preoperative optimization cut-offs at time of diagnosis and those were less likely to undergo hernia repair during the study timeframe compared to those who did (OR 0.50; 95% CI [0.42-0.60]). There was a higher proportion of females (21%) and Blacks (22%) with BMI > 40 kg/m2 compared to males (11%) and other races (11-15%), p = 0.002. As the level of socioeconomic distress increased, there was a corresponding increase in the proportion of patients who did not meet preoperative optimization cut-offs from 16% in prosperous communities to 25% in distressed communities (p < 0.0001). CONCLUSION: Nearly 1 of 5 patients with ventral hernias is affected by commonly used arbitrary preoperative optimization cut-offs. These cut-offs disproportionately impact females, Black patients and those with higher socioeconomic distress. These disparities need to be considered when planning preoperative optimization protocols and resource allocation to ensure equitable access to elective ventral hernia repair.

Crystal structure of the Tspan15 LEL domain reveals a conserved ADAM10 binding site.

Tetraspanins are four-pass transmembrane proteins that function by regulating trafficking of partner proteins and organizing signaling complexes in the membrane. Tspan15, one of a six-member TspanC8 subfamily, forms a complex that regulates the trafficking, maturation, and substrate selectivity of the transmembrane protease ADAM10, an essential enzyme in mammalian physiology that cleaves a wide variety of membrane-anchored substrates, including Notch receptors, amyloid precursor protein, cadherins, and growth factors. We present here crystal structures of the Tspan15 large extracellular loop (LEL) required for functional association with ADAM10 both in isolation and in complex with the Fab fragment of an anti-Tspan15 antibody. Comparison of the Tspan15 LEL with other tetraspanin LEL structures shows that a core helical framework buttresses a variable region that structurally diverges among LELs. Using co-immunoprecipitation and a cellular N-cadherin cleavage assay, we identify a site on Tspan15 required for both ADAM10 binding and promoting substrate cleavage.

Time-resolved phosphoproteomics reveals scaffolding and catalysis-responsive patterns of SHP2-dependent signaling.

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.