Intracellular Ca2+ is normally maintained at submicromolar levels but increases during many forms of cellular stimulation. This increased Ca2+ binds to receptor proteins such as calmodulin (CaM) and alters the cell's metabolism and physiology. Calcium-CaM binds to target proteins and alters their function in such a way as to transduce the Ca2+ signal. Calcium-free or apocalmodulin (ApoCaM) binds to other proteins and has other specific effects. Apocalmodulin has roles in the cell that apparently do not require the ability to bind Ca2+ at all, and these roles appear to be essential for life. Apocalmodulin differs from Ca2+-CaM in its tertiary structure. It binds target proteins differently, utilizing different binding motifs such as the IQ motif and noncontiguous binding sites. Other kinds of binding potentially await discovery. The ApoCaM-binding proteins are a diverse group of at least 15 proteins including enzymes, actin-binding proteins, as well as cytoskeletal and other membrane proteins, including receptors and ion channels. Much of the cellular CaM is bound in a Ca2+-independent manner to membrane structures within the cell, and the proportion bound changes with cell growth and density, suggesting it may be a storage form. Apocalmodulin remains tightly bound to other proteins as subunits and probably hastens the response of these proteins to Ca2+. The overall picture that emerges is that CaM cycles between its Ca2+-bound and Ca2+-free states and in each state binds to different proteins and performs essential functions. Although much of the research focus has been on the roles of Ca2+-CaM, the roles of ApoCaM are equally vital but less well understood.

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M r ϳ68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M r ϳ53,000 and M r ϳ40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAGbinding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the Cterminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/ spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.Extracellular metalloproteases play a pivotal role in the proteolytic processing and turnover of the component molecules of a variety of tissues. Although a number of matrix metalloproteinases (MMPs) 1 may participate in such events, evidence for the involvement of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases in these processes is increasing. For example, ADAMTS-2, ADAMTS-3, and ADAMTS-14 can function as procollagen N-proteinases (1-3), and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease (4 -6).Within the extracellular matrix of cartilage, ADAMTS-4 (7), ADAMTS-5 (8), and ADAMTS-1 (9 -11) may all potentially function as aggrecanases, glutamyl endopeptidases that cleave specific Glu-Xaa bonds of the aggrecan core protein (reviewed in (21)), indicating that regulation of the proteolytic activities of ADAMTS family members is likely to be important for maintenance of homeostasis in a variety of extracellular matrices. Unlike most of the MMPs, which are secreted in a state of latency conferred by the cysteine switch region of the retained propeptide (22), ADAMTS proteases can be cleaved N-terminally by furin or related pro-protein convertase(s) within the trans-Golgi, resulting in secretion of mature, potentially active enzymes lacking the propeptide region. Interestingly, however, ADAMTS family members such as ADAMTS-1 and AD-AMTS-12 have been shown to undergo proteolytic processing within their C-terminal regions, resulting in removal of domains that can bin...

Background-Genetic testing can diagnose long-QT syndrome (LQTS) in asymptomatic relatives of patients with an identified mutation; however, it is costly and subject to availability. The accuracy of a simple algorithm that incorporates resting and exercise ECG parameters for screening LQTS in asymptomatic relatives was evaluated, with genetic testing as the gold standard. Methods and Results-Asymptomatic first-degree relatives of genetically characterized probands were recruited from 5 centers.QT intervals were measured at rest, during exercise, and during recovery. Receiver operating characteristics were used to establish optimal cutoffs. An algorithm for identifying LQTS carriers was developed in a derivation cohort and validated in an independent cohort. The derivation cohort consisted of 69 relatives (28 with LQT1, 20 with LQT2, and 21 noncarriers).Mean age was 35Ϯ18 years, and resting corrected QT interval (QTc) was 466Ϯ39 ms. Abnormal resting QTc (females Ն480 ms; males Ն470 ms) was 100% specific for gene carrier status, but was observed in only 48% of patients; however, mutations were observed in 68% and 42% of patients with a borderline or normal resting QTc, respectively. Among these patients, 4-minute recovery QTc Ն445 ms correctly restratified 22 of 25 patients as having LQTS and 19 of 21 patients as being noncarriers. The combination of resting and 4-minute recovery QTc in a screening algorithm yielded a sensitivity of 0.94 and specificity of 0.90 for detecting LQTS carriers. When applied to the validation cohort (nϭ152; 58 with LQT1, 61 with LQT2, and 33 noncarriers; QTcϭ443Ϯ47 ms), sensitivity was 0.92 and specificity was 0.82. Conclusions-A simple algorithm that incorporates resting and exercise-recovery QTc is useful in identifying LQTS in asymptomatic relatives. (Circulation. 2011;124:2187-2194.)