Browsing by Subject "Intracellular Signaling Peptides and Proteins"
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Item Calpain 9 functions in TNF receptor mediated apoptosis(2012-07-20) Kunkel, Gregory Thomas; Wang, XiadongEvasion of apoptosis is a hallmark of cancer development. The Inhibitor of Apoptosis Proteins, IAPs, block Caspase activity and cell death. Release of the Second Mitochondria-Derived Activator of Caspases, Smac, from the mitochondria relieves IAP Caspase inhibition, activating apoptosis. Our lab has developed a small molecule Smac mimetic. Surprisingly, approximately 25% of cell lines show single agent Smac mimetic sensitivity through activation of autocrine TNF-a secretion and TNF dependent apoptosis. Using Smac mimetic sensitivity as a model system, I performed a genome-wide high-throughput siRNA screen and identified Calpain 9, CAPN9, as a novel component of TNF-alpha induced apoptosis. CAPN9 knockdown does not affect TNF-a secretion, yet is essential for downstream activation. Two splice variants are reported for CAPN9. The smaller splice, CAPN9-SP2, is required for effective TNF-a induced apoptosis. CAPN9 is essential for RIPK1 recruitment and ubiquitination at the TNFR1 upon activation with TNF-alpha. CAPN9 knockdown demonstrates previously unreported association of ubiquitinated proteins, and actin binding proteins with TNFR1 in the absence of stimulus. This interaction is CAPN9 dependent and correlates with CAPN9 regulation of TNF-a induced apoptosis. [Keywords: Calpain, SMAC, IAR, TNF, ubiquitin, RJPK1]Item Delineating the Role of CD244 in NK Cell Cytotoxicity and the Contribution of Ly108 to Thymocyte Development(2010-11-02T18:20:55Z) Westcott, Jill Michelle; Schatzle, JohnGenetic analysis of the a murine model of lupus has implicated polymorphisms in the SLAM family of receptors (CD244 (2B4), CD229 (Ly9), CS-1 (CRACC), CD48 CD150 (SLAM), CD84, and Ly108 (NTB-A)) as causative for a breach in tolerance of both T and B cells leading to the production of self reactive antibodies. Analysis of common strains of laboratory mice revealed the existence of two stable haplotypes (b and z) of the SLAM family gene cluster. Given recent studies identifying polymorphisms in the SLAM family in humans, we have determined how polymorphisms in the SLAM family can affect lymphocyte function in mice to model how similar mechanisms may be involved in human pathologies. For these studies, we used mice congenic at the SLAM family locus (B6 –b haplotype and B6.Sle1b–z haplotype) to study two major questions: 1.) how do polymorphisms in CD244 affect NK function? And 2.) how do polymorphisms in Ly108 affect T cell tolerance? In the studies presented herein we demonstrate that CD244 functions largely as an inhibitory receptor in NK cells from B6 mice and as an activating receptor in B6.Sle1b mice. We demonstrate that allelic polymorphisms contribute to this differential function by altering receptor isoform usage, cell surface densities, baseline phosphorylation levels and subsequent adaptor association and receptor downmodulation. We also demonstrate that differential isoform usage of the receptor, Ly108 in B6 and B6.Sle1b mice alters thymocyte differentiation and negative selection events leading to a break in tolerance of T cells in B6.Sle1b mice. This is due to differential affects of the isoforms of Ly108 on thymocyte cell cycle progression and sensitivity to apoptosis. These studies highlight how naturally occurring polymorphisms in SLAM family genes can profoundly affect receptor function and potentially result in pathologic outcomes in certain genetic contexts.Item Evolutionary Constraints Specifying Protein Folding and Function(2007-08-04) Larson, Christopher; Ranganathan, RamaProteins are complex macromolecules that carry out biological functions while under constant mutational load and selective pressure during evolution. Consequently, evolution has generated protein families by exploring the set of sequences able to carry out a particular biological activity, maintaining sequence motifs critical for function while varying the rest of the protein. Statistical coupling analysis of a protein family examines an alignment of such sequences and detects the evolutionarily preserved interresidue interactions critical for the proteins' selective fitness. This set of information has provided a sufficiently detailed description of evolutionary design constraints to allow the design of novel WW domain sequences that fold and function like natural proteins. This work expands the initial investigations, and probes the minimal information content necessary to specify the WW domain fold, as well as the effect of increasing the coupling constraints in the design process. This work also evaluates the ability of coupling information to design larger and more complex protein folds and to specify their biological functions. Experimental expression and characterization of WW domains designed with varying levels of coupling information indicates that incorporating even small amounts of coupling information has a notable impact on these proteins' ability to fold. Moreover, different coupling-based design approaches produce results robust to details of how coupling information is incorporated. Similar experiments with designed PDZ domains and in vivo characterization of designed G-protein coupled receptors show that, to the extent studied, this design approach is successful with these larger and more complex proteins as well. This indicates that the typically sparse matrices of coupling values observed for a protein family capture the core evolutionary constraints on the proteins in sufficient detail to generate even complex proteins with natural-like folds and functions.Item The Function of the TCR zeta zeta Module in T Cells(2005-04-29) Pitcher, Lisa Anne; Oers, Nicolai vanThe alpha beta T cell receptor complex (TCR) has the unique ability to discriminate and differentially respond to peptide/MHC ligands encountered on the surface of antigen presenting cells. The ligation of the TCR with peptide/MHC complexes is translated into intracellular signals through a conserved sequence motif, termed ITAM, or immunoreceptor tyrosine-based activation motif, which are present in one or more copies in the cytoplasmic portions of the TCR zeta and CD3 gamma, delta and epsilon chains. A distinctive feature of the TCR complex is that it contains ten ITAMs, in contrast to other antigen receptor complexes which contain two or four ITAMs. The ten TCR ITAMs are distributed as one in each CD3 chain and three in the TCR zeta subunit (TCR alpha beta epsilon delta epsilon gamma zeta zeta). It has been proposed that the TCR is comprised of two autonomous signaling modules, TCR zeta zeta and CD3 gamma epsilon/delta epsilon. Following receptor ligation, TCR zeta is the most heavily tyrosine-phosphorylated subunit of the TCR, developing into two stable intermediates of 21- and 23-kDa (p21 and p23). Based on the number of ITAMs it contributes, TCR zeta zeta was initially presumed to be the predominant signaling module in the TCR complex, with p21 and p23 being linked to virtually all aspects of T cell biology. To clearly define the functions of the TCR zeta zeta module, we generated a series of TCR zeta transgenic mice, with modified zeta molecules, that selectively express p21 alone, no p21 or p23, or no phospho-zeta intermediates. In a wild type or high affinity TCR system (P14), T cell development was completely normal in the TCR zeta transgenic lines. Surprisingly, when peripheral T cells were analyzed for their functionality in response to various stimuli, including peptide/MHC-, T cell mitogen- and superantigenic- stimulation, equivalent dose response curves were observed, regardless of phospho-zeta. Notably, these data also eliminated a possible inhibitory role for the partially phosphorylated p21 intermediate of TCR zeta. To more carefully examine the roles of p21 and p23, the TCR zeta transgenics were mated to a TCR transgenic line (HY) bearing T cells with a low affinity TCR. In this system, important roles for phospho-zeta during T cell selection were revealed. Specifically, TCR zeta ITAMs functioned additively during positive selection events. These selection events appear to be independent of traditional signaling pathways, as the signaling capacity of unselected T cells in the absence of all phospho-zeta was equivalent to T cells with wild-type TCR zeta subunits. These results imply that the CD3 gamma epsilon/delta epsilon module is the predominant signaling module in the TCR complex. Our studies also identified a unique role for p21 during negative selection events. The select expression of p21 in T cells attenuated negative selection of thymocytes, resulting in the generation of a population of potentially autoreactive cells. Based on these data, a revised model of TCR signal transmission is proposed. Within this model, the TCR zeta zeta and CD3 gamma epsilon/delta epsilon modules contribute both redundant and unique functions to T cells. The CD3 gamma epsilon/delta epsilon module is primarily responsible for classical TCR-mediated signaling pathways leading to T cell activation. The TCR zeta zeta and CD3 gamma epsilon/delta epsilon modules contribute redundant functions to thymocyte positive selection. These redundant functions are mediated by the ten TCR ITAMs. Phosphorylated intermediates of the TCR zeta zeta module also contribute to thymocyte positive selection, likely through alternative signaling pathways. In addition, the TCR zeta zeta module functions in a unique manner during thymocyte negative selection, with p21 attenuating negative selection of thymocytes. Furthermore, preliminary evidence suggests novel roles for the TCR zeta zeta module in the maintenance of peripheral T cells and in the adaptive immune response to bacterial pathogens.Item The Hippo Signaling Pathway in Organ Size Control and Regeneraton(2012-07-17) Ren, Fangfang; Jiang, JinThe Hippo (Hpo) signaling pathway controls cell growth, proliferation and apoptosis in both Drosophila and vertebrates. Our lab has previously demonstrated that Hpo signaling regulates gene expression by inhibiting a transcription complex consisting of the transcriptional coactivator Yorkie (Yki) and the TEAD/TEF family of transcription factor Scalloped (Sd) in Drosophila. The inhibition of Yki activity is through modulating its phosphorylation status and subcellular localization by upstream kinase complex. I obtained both genetic and cellular evidence that 14-3-3 proteins are involved in this process. I also identified three Serine residues (S111, S168 and S250 of Yki as essential for restricting Yki activity. I found that 14-3-3 regulates Yki subcellular localization mainly through S168 but not the other two sites. The recent identification of intestinal stem cells (ISCs) has made the Drosophila adult midgut an excellent model to study adult stem cell biology. Multiple signaling pathways have been implicated in the regulation of ISC proliferation, self-renewal and differentiation. I obtained evidence that Hpo signaling plays an essential role in regulating ISC proliferation through both cell-autonomous and non-cell-autonomous mechanisms. Cytokines of the Upd family and multiple EGFR ligands were found to be ectopically induced when Hpo signaling is inactivated in differentiated cells, which in turn activate Jak-Stat and EGFR signaling pathways in ISCs to stimulate their proliferation. I also showed that tissue damaging reagent DSS-induced ISC proliferation is dependent on Yki activity in precursor cells. Although several signaling pathways including Jak-Stat, EGFR and Hpo pathways have been implicated in damage-induced ISC proliferation, the cell intrinsic mechanisms have remained elusive. I found that the Drosophila homolog of Myc oncogene (dMyc), which encodes a transcription regulator that affects cellular growth and cell cycle progression, functions downstream of Hpo, Jak-Stat and EGFR pathways to mediate their effects on ISC proliferation. dMyc is also essential for adult midgut homeostasis as well as regeneration after exposure to damage reagents. I also demonstrated that the regulation of dMyc levels by Hipo, Jak-Stat and EGFR pathways is at the level of transcription. [Keywords: Drosphila, hippo, Yki, instestine stem cell, regeneration]Item Insig-Mediated Regulation of Hepatic Lipid Synthesis(2007-05-22) Engelking, Luke James; Brown, Michael S.Cholesterol synthesis in mammals is tightly regulated by end-product feedback inhibition. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a rate determining reaction that is highly regulated by transcriptional and post-transcriptional mechanisms. As cellular cholesterol accumulates, the transcription of HMGR mRNA is suppressed and the proteosomal degradation of HMGR protein is accelerated. The sterol-regulated transcription of HMGR and other lipogenic genes is controlled by sterol regulatory element binding proteins (SREBPs). These membrane-bound transcription factors are escorted by SREBP cleavage activating protein (SCAP) from the endoplasmic reticulum (ER) to the Golgi apparatus where SREBPs are proteolytically processed to their active forms. In cultured cells, feedback inhibition of SREBP processing is mediated by Insigs. When sterols accumulate, Insigs block SREBP activation by retaining SCAP in the ER. Insigs also mediate rapid, sterol-dependent turnover of HMGR protein. When sterols accumulate, Insigs bind to HMGR and stimulate its ubiquitination and degradation. Although Insigs are key regulators of cholesterol homeostasis in cultured cells, their role in the intact mammal was undefined. To explore this question, gain-of-function and loss-of-function analyses were performed by studying the livers of genetically engineered mice. First, transgenic mice that overexpress Insig-1 in liver (TgInsig-1) were generated. In the livers of TgInsig-1 mice, nuclear SREBPs (nSREBPs) were reduced and SREBP processing was supersensitive to inhibition by feeding high-cholesterol diets. The block in SREBP processing reduced the mRNA levels of SREBP target genes. Levels of HMGR protein were reduced and declined further with cholesterol feeding. Next, knockout mice that lack Insig-1, Insig-2, or both Insigs were generated. In the livers of Insig double knockout mice, cholesterol and triglycerides accumulated to high levels, and despite their accumulation, nSREBPs and mRNAs of SREBP target genes were not suppressed. SREBP processing was insensitive to inhibition by feeding high-cholesterol diets. HMGR protein levels were increased and failed to decline with cholesterol feeding. As a consequence of Insig overexpression or deficiency and the respective effect on SREBPs and HMGR, hepatic cholesterol and fatty acid synthesis in living animals was decreased in TgInsig-1 mice and increased in Insig double knockout mice. These studies indicate that Insigs are essential regulators of hepatic lipid synthesis.Item The Intracellular Domain of EPHB1 Is Required For Axon Pathfinding at the Optic Chiasm and Corpus Callosum(2011-08-26T17:34:18Z) Chenaux, George; Henkemeyer, MarkThis dissertation presents evidence of the importance of EphB1 mediated signaling in retinal and callosal axons while attempting to reach their targets. EphB receptor tyrosine kinases direct axonal pathfinding through interactions with ephrin-B proteins following axon-cell contact. Since EphB:ephrin-B binding leads to bidirectional signals, the contributions of signaling into the Eph-expressing cell (forward signaling) or the ephrin-expressing cell (reverse signaling) cannot be assigned using traditional protein-null alleles. To determine if EphB1 is functioning as a receptor during axon pathfinding, I created a new knock-in mutant mouse, EphB1 T-lacZ, that expresses an intracellular-truncated EphB1-β-gal fusion protein from the endogenous locus. As in the EphB1 -/-protein-null animals, the EphB1 T-lacZ/T-lacZ homozygotes fail to form the ipsilateral projecting subpopulation of retinal ganglion cell (RGC) axons. This indicates that forward signaling through the intracellular domain of EphB1 is required for proper axon pathfinding of RGC axons at the optic chiasm. Further analysis of other EphB and ephrin-B mutant mice shows that EphB1 is the preferred receptor of both ephrin-B1 and ephrin-B2 in mediating axon guidance at the optic chiasm despite the coexpression of EphB2 in the same ipsilaterally projecting RGC axons. In addition to analyzing the axon pathfinding defect at the optic chiasm, the EphB1 T-lacZ mice were also used to analyze another phenotype associated with EphB1 -/-protein-null animals, a failure to properly form a corpus callosum. I will show that the intracellular domains of EphB1 and EphB2 are important for the guidance of callosal axons across the midline during the formation of the corpus callosum. However, opposite to the above mentioned optic chiasm phenotype, these animals have axons that fail to project contralaterally choosing to remain on the ipsilateral hemisphere.Item Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap(2011-12-14) Motamed, Massoud; Brown, Michael S.Low density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes, allowing cholesterol to be released. Export of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two lysosomal proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, a membrane protein with 13 putative membrane-spanning helices. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death owing to lipid accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind to cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1 (NTD), can be expressed in vitro as a soluble protein of 240 amino acids that maintains cholesterol binding activity. This thesis studies NPC2 in detail as summarized below. Despite a shared role as cholesterol binding proteins, NPC2 and NPC1 (NTD) bind to cholesterol in opposite orientations. The crystal structures of NPC2 and NPC1 (NTD) have been solved, and NPC2 binds cholesterol with the iso-octyl chain facing the interior of the protein, whereas, NPC1(NTD) binds cholesterol with the 3ß-hydroxyl facing the interior of the protein. Another striking difference is the kinetics of this cholesterol binding. NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1 (NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC). However, NPC2 can stimulate the rate of cholesterol binding to NPC1 (NTD) (>15-fold in vitro). This stimulation of cholesterol binding to NPC1 (NTD) by NPC2 is believed to occur through a direct transfer of cholesterol from NPC2 to NPC1(NTD). Amino acid residues important for binding or transfer of cholesterol on NPC2 were identified through alanine scan mutagenesis. Residues that decreased binding thermodynamics and/or kinetics mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the exposed residues of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most deleterious binding mutant was P120S, a residue in the cholesterol binding pocket; the most deleterious transfer mutant was V81D, a residue on the hydrophobic patch extending outward from the cholesterol binding pocket. The above mutants of NPC2 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2-deficient cells, in contrast to wild-type NPC2. Once LDL-derived cholesterol leaves the lysosomes, it is transported to the endoplasmic reticulum (ER), where it serves a regulatory role in cholesterol homeostasis. In the ER, these regulatory functions include activation of acetyl-coenzyme A acetyltransferase (ACAT), allowing for esterification of cholesterol for storage, and regulation of sterol regulatory element–binding protein (SREBP) localization, a transcription factor that regulates key enzymes for cholesterol synthesis. SREBP cleavage-activating protein (Scap) is the switch that controls SREBP, and therefore cholesterol synthesis. Scap senses cholesterol abundance in the ER and acts as an escort protein. In sterol depleted cells, Scap escorts SREBP to the Golgi complex, where two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can go to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. When cholesterol in abundant, the sterol binds to Scap and triggers a conformational change in the protein that prevents it from escorting SREBPs to the Golgi for proteolytic cleavage. Scap is a 1276 amino acid protein that consists of two domains: an N-terminal domain with 8 transmembrane spanning regions and a C-terminal domain that projects into the cytosol and associates with SREBPs. Previous studies have localized the cholesterol-binding activity of Scap to its membrane domain. Studies described in this thesis identify the cholesterol binding pocket in Scap and identify key residues that play an important role in the protein’s responsiveness to cholesterol binding. The first loop region of Scap (hereafter referred to as Scap(Loop1)) was purified as a recombinant protein and found to have cholesterol binding activity. The specificity of this sterol binding was determined through competition studies and shown to be physiologically relevant. Additionally, this binding affinity and specificity was similar to that of the membrane domain of Scap. Subsequently, alanine scan mutagenesis was performed on Scap(Loop1). Through this approach, several mutations of Scap were identified that constitutively adopt the cholesterol-bound state. This data demonstrates that Scap(Loop1) binds to cholesterol and that the binding then helps induce the conformational change required for Scap to anchor SREBP in ER membranes.Item Orexin Signaling and the Prevention of Diet-Induced Obesity(2012-08-13) Tsai, Allen Lee; Yanagisawa, MasashiThe hypothalamic neuropeptide orexin acutely promotes appetite, yet orexin deficiency in humans and mice is associated with obesity. Prolonged effects of orexin signaling upon energy homeostasis have not been fully characterized. In this study, I utilized both genetic and pharmacologic approaches to characterize metabolic effects of orexin gain of function. CAG/orexin transgenic mice confer resistance to high-fat diet-induced obesity and insulin insensitivity by promoting energy expenditure and reducing food consumption. Genetic studies indicated that orexin receptor-2 (OX2R), rather than orexin receptor-1 (OX1R) signaling, predominantly mediates this phenotype. Likewise, prolonged central infusion of an OX2R selective peptide agonist prevents diet-induced obesity. While orexin overexpression enhances the anorectic-catabolic effects of central leptin administration, obese leptin-deficient mice (ob/ob) are completely resistant to the metabolic effects of orexin overexpression or OX2R selective agonist administration. I conclude that enhanced orexin-OX2R signaling confers resistance to diet-induced features of the metabolic syndrome through promoting a negative energy homeostasis and improving leptin sensitivity. [Keywords: orexin, obesity, high-fat diet, leptin, hypothalamus, intracerobroventricular, central, insulin, orexin receptor 2]Item SCAP, Insig, and Cholesterol Interactions in Mammalian Cells(2007-05-22) Feramisco, Jamison Derek; Brown, Michael S.Cholesterol synthesis in mammalian cells is highly regulated by an end-product feedback mechanism. The transcription of genes necessary for both fatty acid and cholesterol production are controlled by sterol regulatory element binding proteins (SREBPs). The critical regulatory step is the proteolytic release of SREBPs from their inactive membrane bound form. Soon after translation, SREBPs bind SREBP cleavage activating protein (SCAP), a polytopic membrane protein of the endoplasmic reticulum (ER). In sterol depleted situations, SCAP escorts SREBPs to the Golgi, where SREBPs are cleaved and can move freely to the nucleus and activate the numerous enzymes of cellular lipid homeostasis. When cellular sterol levels rise, the SCAP/SREBP complex binds to an ER resident protein named Insig. Upon binding to Insig, the movement of the SCAP/SREBP complex to the Golgi is inhibited, thus halting cholesterol and fatty acid synthesis. The mechanism by which the cell senses sterol levels has been long unknown. Radhakrishnan et al. and Adams et al. demonstrated that SCAP itself binds cholesterol and thus may act directly to sense cellular sterol levels and mediate the end-product feedback control of SREBPs. The goal of this thesis is to elucidate the molecular details of the interactions between SCAP, Insig and cholesterol. My thesis experimentally details the membrane topology of human Insig-1 and shows that it is a polytopic integral membrane protein of the ER with six transmembrane spanning segments. In addition, the amino and carboxy-termini of Insig are both facing the cytosol. Furthermore, crucial residues of Insig that are important for SCAP interaction are identified. My thesis has also defined distinct amino acids of SCAP that are essential for its role as a protein that binds Insig and as a protein that has the ability to bind cholesterol. An aspartic acid in the middle of transmembrane six is necessary for sterol regulated binding to Inisg, while residues in transmembrane segments one and three of SCAP are crucial for cholesterol binding both in vivo and in vitro.Item A WNK and a Nudge towards Kinase Biology(2013-01-17) Sengupta, Samarpita; Cobb, Melanie H., Ph.D.A family of four atypical protein kinases; WNKs are characterized by a non-canonical position of the catalytic lysine. The significance of WNKs was first realized when they were found to be causatively linked to a rare form of genetic hypertensive disease known as PHAII. WNKs are an ancient family of kinases and are known to have roles in regulating salt homeostasis in the body in response to osmotic stress, regulating vesicular transport and regulating circadian rhythm in plants. All four WNKs can activate their downstream substrate, OSR1 which in turn can activate ion cotransporters downstream such as NCC and NKCC which results in regulation of ion balance in the cell. WNKs can bind to each other and can potentially form an autoactivable complex. Thus, regulation by WNKs is a complex affair. Further, OSR1 binds to its upstream regulators and substrates via RFxV motifs. WNK1, depending on the splice form, possesses at least five RFxV motifs. I have defined a minimal region on WNK1 required for interaction with OSR1. I have shown that expression of this minimal binding region in cells is sufficient to inhibit OSR1 activation by WNKs. I have also determined that the WNK-OSR1 pathway can cross-talk with the mTORC2 pathway. mTORC2 can directly phosphorylate OSR1 and regulate its activity. Finally, to understand why WNKs possess a slow substrate turnover rate and whether a crucial cofactor is missing, I have determined that WNKs can bind to lipids in vitro. Lipids can alter kinase activity of WNK1 towards OSR1. Future studies will be aimed at understanding the mechanism of action of mTORC2 in regulating the WNK-OSR1 pathway and to determine whether WNKs can bind to lipids in cells and the importance of the lipid binding activity of WNKs. Understanding the intricacies of WNKs would give us important tools to determine its roles in human diseases.