Faculty Bibliography

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca²⁺-binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential.Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942-52. ©2017 AACR.

Fatty liver is the most common type of liver disease, affecting nearly one third of the US population and more than half a billion people worldwide. Abnormalities in ER calcium handling and mitochondrial function each have been implicated in abnormal lipid droplet formation. Here we show that the type 1 isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R1) specifically links ER calcium release to mitochondrial calcium signaling and lipid droplet formation in hepatocytes. Moreover, liver-specific InsP₃R1 knockout mice have impaired mitochondrial calcium signaling, decreased hepatic triglycerides, reduced lipid droplet formation and are resistant to development of fatty liver. Patients with non-alcoholic steatohepatitis, the most malignant form of fatty liver, have increased hepatic expression of InsP₃R1 and the extent of ER-mitochondrial co-localization correlates with the degree of steatosis in human liver biopsies.

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca²⁺ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O₂ levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O₂-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O₂ and show less mitochondrial Ca²⁺ uptake in response to bradykinin-induced ER Ca²⁺ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O₂ levels.

Noonan syndrome (NS) is a common autosomal dominant disorder that presents with short stature, craniofacial dysmorphism, and cardiac abnormalities. Activating mutations in the PTPN11 gene encoding for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) causes approximately 50% of NS cases. In contrast, NS with multiple lentigines (NSML) is caused by mutations that inactivate SHP2, but it exhibits some overlapping abnormalities with NS. Protein zero-related (PZR) is a SHP2-binding protein that is hyper-tyrosyl phosphorylated in the hearts of mice from NS and NSML, suggesting that PZR and the tyrosine kinase that catalyzes its phosphorylation represent common targets for these diseases. We show that the tyrosine kinase inhibitor, dasatinib, at doses orders of magnitude lower than that used for its anticancer activities inhibited PZR tyrosyl phosphorylation in the hearts of NS mice. Low-dose dasatinib treatment of NS mice markedly improved cardiomyocyte contractility and functionality. Remarkably, a low dose of dasatinib reversed the expression levels of molecular markers of cardiomyopathy and reduced cardiac fibrosis in NS and NSML mice. These results suggest that PZR/SHP2 signaling is a common target of both NS and NSML and that low-dose dasatinib may represent a unifying therapy for the treatment of PTPN11-related cardiomyopathies.

Mutations in polycystin-2 (PC2) lead to autosomal dominant polycystic kidney disease (ADPKD). The molecular mechanism linking mutations in PC2 and the pathogenesis of ADPKD is not well understood. Therefore, understanding the functional regulation of PC2 and its interaction with other proteins under both physiological and pathogenic conditions is important for elucidating the disease mechanism and identifying potential molecular targets for treatment. Normally, PC2 functions as a calcium-permeable channel whose activity is regulated by calcium binding to the C-terminal domain of PC2 (PC2 Cterm). The PC2 Cterm is also involved in the PC2 channel assembly and hetero-oligomerization with other binding partners in cells. Different functional domains of the PC2 Cterm have been studied using structural approaches. Within the PC2 Cterm, there is a calcium-binding EF-hand domain, crucial for the calcium-dependent activity of the PC2 channel. Downstream of the EF-hand domain lies a coiled-coil region, which is involved in the assembly and hetero-interaction of the PC2 protein. The PC2 Cterm can form an oligomer, mediated by the coiled-coil region. Although PC2 Cterm has been extensively studied for its relationship with ADPKD and its importance in PC2 regulation, there are misunderstandings with respect to the definition of the domain topology within the PC2 Cterm and the functional role of each domain. Here, we review previous studies that connect the molecular properties of the domains of PC2 Cterm to distinct aspects of PC2 functions and regulation.

PC2 (polycystin-2) forms a Ca(2+)-permeable channel in the cell membrane and its function is regulated by cytosolic Ca(2+) levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca(2+)-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca(2+) regulates the channel through the HPC2 Cterm, we characterized Ca(2+) binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca(2+). The HPC2 L1EF construct contains the Ca(2+)-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca(2+)-bound conformation of the EF-hand, thus enhancing its Ca(2+)-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca(2+), we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca(2+) binding. Nevertheless, they have different profiles of H-D exchange under non-saturating Ca(2+) conditions, implying their different conformational exchange between the Ca(2+)-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca(2+)-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease.

Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We examined a poorly characterized variant of NCS-1 (Var2), identified only in humans where the N-terminal 22 amino acid residues of native NCS-1(MGKSNSKLKPEVVEELTRKTY) were replaced with 4 different residues (MATI). Because alterations in the level of expression of NCS-1 Var1 and the expression of NCS-1 variants have been correlated with several neurological diseases, the relative expression and functional role of NCS-1 Var2 was examined. We found that NCS-1 Var2 mRNA levels are not found in mouse tissues and are expressed at levels ~1000-fold lower than NCS-1 Var1 in three different human cell lines (SHSY5Y, HEK293, MB231). Protein expression of both variants was only identified in cell lines overexpressing exogenous NCS-1 Var2. The calcium binding affinity is ~100 times weaker in purified NCS-1 Var2 than NCS-1 Var1. Because truncation of NCS-1 Var1 has been linked to functional changes in neurons, we determined whether the differing properties of the NCS-1 variants could potentially contribute to the altered cell function. In contrast to previous reports showing that overexpression of NCS-1 Var1 increases calcium-dependent processes, functional differences in cells overexpressing NCS-1 Var2 were undetectable in assays for cell growth, cell death and drug (paclitaxel) potency. Our results suggest that NCS-1 Var1 is the primary functional version of NCS-1.

Mutations in the gene for polycystin 2 (Pkd2) lead to polycystic kidney disease, however the main cause of mortality in humans is cardiac related. We previously showed that 5 month old Pkd2+/- mice have altered calcium-contractile activity in cardiomyocytes, but have preserved cardiac function. Here, we examined 1 and 9 month old Pkd2+/- mice to determine if decreased amounts of functional polycystin 2 leads to impaired cardiac function with aging. We observed changes in calcium handling proteins in 1 month old Pkd2+/- mice, and these changes were exacerbated in 9 month old Pkd2+/- mice. Anatomically, the 9 month old Pkd2+/- mice had thinner left ventricular walls, consistent with dilated cardiomyopathy, and the left ventricular ejection fraction was decreased. Intriguingly, in response to acute isoproterenol stimulation to examine β-adrenergic responses, the 9 month old Pkd2+/- mice exhibited a stronger contractile response, which also coincided with preserved localization of the β2 adrenergic receptor. Importantly, the Pkd2+/- mice did not have any renal impairment. We conclude that the cardiac-related impact of decreased polycystin 2 progresses over time towards cardiac dysfunction and altered adrenergic signaling. These results provide further evidence that polycystin 2 provides a critical function in the heart, independent of renal involvement.

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

The GxGD proteases function to cleave protein substrates within the membrane. As these proteases contain multiple transmembrane domains typical of ion channels, we examined if GxGD proteases also function as ion channels. We tested the putative dual function by examining two archeobacterial GxGD proteases (PSH and FlaK), with known three-dimensional structures. Both are in the same GxGD family as presenilin, a protein mutated in Alzheimer Disease. Here, we demonstrate that PSH and FlaK form cation channels in lipid bilayers. A mutation that affected the enzymatic activity of FlaK rendered the channel catalytically inactive and altered the ion selectivity, indicating that the ion channel and the catalytic activities are linked. We report that the GxGD proteases, PSH and FlaK, are true "chanzymes" with interdependent ion channel and protease activity conferred by a single structural domain embedded in the membrane, supporting the proposal that higher-order proteases, including presenilin, have channel function.