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Calreticulin (CRT) is an intrinsically disordered multifunctional protein that plays essential roles intra-and extra-cellularly. The Michalak laboratory has proposed that CRT was initially identified in 1974 by the MacLennan laboratory as the high-affinity Ca2+-binding protein (HACBP) of the sarcoplasmic reticulin (SR). This widely accepted belief has been ingrained in the scientific literature but has never been rigorously tested. In our report, we have undertaken a comprehensive reexamination of this assumption by meticulously examining the majority of published studies that present a proteomic analysis of the SR. These analyses have utilized proteomic analysis of purified SR preparations or purified components of the SR, namely the longitudinal tubules and junctional terminal cisternae. These studies have consistently failed to detect the HACBP or CRT in skeletal muscle SR. We propose that the existence of the HACBP has failed the test of reproducibility and should be retired to the annals of antiquity. Therefore, the scientific dogma that the HACBP and CRT are identical proteins is a non sequitur. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple‐negative breast cancer (TNBC) progression has been linked to ALDH1A3‐induced gene expression changes. To investigate the mechanism of ALDH1A3‐mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all‐trans‐retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen‐dependent. In patient tumours, ALDH1A3 and tPA are co‐expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3–tPA–plasmin axis as a key contributor to breast cancer progression. [ABSTRACT FROM AUTHOR]

Hyaluronidases are a family of endolytic glycoside hydrolases that cleave the β1-4 linkage between N-acetylglucosamine and glucuronic acid in hyaluronan polymers via a substrate-assisted mechanism. In humans, turnover of hyaluronan by this enzyme family is critical for normal extracellular matrix remodeling. However, elevated expression of the Hyall isozyme accelerates tumor growth and metastatic progression. In this study, we used structural information, site-directed mutagenesis, and steady state enzyme kinetics to probe molecular determinants of human Hyall function. Mutagenesis of active site residues Glu[sup131] and Tyr[sup247] to Gln and Phe, respectively, eliminated activity at all hyaluronan concentrations (to 125 μM or 2.5 mg/ml). Conservative mutagenesis of Asp[sup129] and Tyr[sup202] significantly impaired catalysis by increases of 5and 10-fold in apparent K[subm], and reductions in Vmax of 95 and 50%, respectively. Tyr[sup247] and Asp[sup129] are required for stabilization of the catalytic nucleophile, which arises as a resonance intermediate of N-acetylglucosamine on the substrate. Glu[sup131] is a likely proton donor for the hydroxyl leaving group. Tyr[sup202] is a substrate binding determinant. General disulfide reduction had no effect on activity in solution, but enzymatic deglycosylation reduced Hyall activity in a time-dependent fashion. Mutagenesis identified Asn[sup350] glycosylation as the requisite modification. Deletion of the C-terminal epidermal growth factor-like domain, in which Asn[sup350] is located, also eliminated activity, irrespective of glycosylation. Collectively, these studies define key components of Hyall active site catalysis, and structural factors critical for stability. Such detailed understanding will allow rational design of enzyme modulators. [ABSTRACT FROM AUTHOR]