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The transcriptional factor, Tead1, mediates the transcriptional output of the Hippo pathway. Tead1 activates the transcription of downstream genes including many cell cycle promoting genes that lead to cell proliferation in many tissues, but its role in β-cells is unknown. To test if Tead1 is required for β-cell proliferation and function, we deleted Tead1 in β-cell. Deletion of Tead1 in β-cells was confirmed in these β-cell Tead1 KO (β-Tead1-/-) , by a significant decrease in transcript and protein from whole islet lysates. The β-Tead1-/- mice had normal body weight but developed significantly higher fasting and fed glucose levels starting at 5 weeks of age and progressed to frank diabetes by 8 weeks, with fasting blood glucose >300 mg/dl, accompanied by hypoinsulinemia, as compared to floxed control mice. 8wk β-Tead1-/- mice display severe glucose intolerance due to an abrogation of GSIS during GTT, with a loss of first phase insulin secretion. Pancreatic insulin content was decreased by ∼50%, due to a decrease in expression of mature β-cell genes including, Pdx1, Nkx6.1 and MafA, all of which, we show to be direct transcriptional targets of Tead1. Tead1 also binds to the proximal promoter at the Ink4a locus to regulate p16/p19, critical cell cycle inhibitors, and this loss of activation in Tead1-deficient β-cells leads to an enhanced entry into cell cycle. Furthermore, to test if Tead1 has a similar regulatory role in human β-cells, we deleted TEAD1 knockout in human iPS cell-derived β-cells using CRISPR/Cas9 system. Tead1 deficient iPS cell-derived β-cells displayed a significant decrease in vitro GSIS. Our results demonstrate Tead1 to be a transcriptional switch required for β-cells to maintain mature function and remain in proliferative quiescence. This novel regulatory pathway could be targeted for β-cell replacement therapy to achieve β-cell proliferation without a loss of mature function. Disclosure J. Lee: None. V. Yechoor: n/a. R. Liu: None. F. Li: None. V. Negi: None. R. Jagannathan: None. M. Huising: Consultant; Crinetics Pharmaceuticals, Inc., Research Support; Crinetics Pharmaceuticals, Inc. K. Ma: None. B. Shih: None. M. Moulik: None. Funding VA-MERIT Grant (I01BX002678)

Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that Tead1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function, and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, although its role in cardiomyocytes is unknown. Here, we show that Tead1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle, and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss of function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that Tead1 regulation of OXPHOS in cardiomyocytes is cell autonomous. Taken together, our data demonstrate that Tead1 is a crucial transcriptional node that is a cell-autonomous regulator, a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis. NEW & NOTEWORTHY Mitochondrial dysfunction constitutes an important aspect of heart failure etiopathogenesis and progression. However, the molecular mechanisms are still largely unknown. Growing lines of evidence indicate that Hippo-Tead pathway plays a role in cellular bioenergetics. This study reveals the novel role of Tead1, the downstream transcriptional effector of Hippo pathway, as a novel regulator of mitochondrial oxidative phosphorylation and in vivo cardiomyocyte energy metabolism, thus providing a potential therapeutic target for modulating mitochondrial function and enhancing cytoprotection of cardiomyocytes.

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.

Introduction: Mammalian cardiomyocyte (CM) proliferation peaks in the embryonic and neonatal periods. TEAD1, a key transcription factor regulated by the Hippo pathway, is critical for early embryonic CM proliferation. But mid gestation lethality of Tead1 germline deletion precluded the study of its role in CMs at later developmental stages. We recently generated Tead1 floxed (Tead1 F/F ) mice which allows the study of TEAD1 function in CMs at later stages. The objective of this study was to determine requirement of TEAD1 for neonatal CM proliferation. Hypothesis: TEAD1 remains critical for CM proliferation in late embryonic and early neonatal periods through transcriptional regulation of cell cycle promoting genes. Methods and Results: We observed that TEAD1 cardiac expression peaks in the perinatal period. Using Myh6-Cre deletor mice, we knocked out Tead1 in CMs at E10.5 (referred as cKO). cKO pups were born in expected Mendelian frequency, but survived only till day of life (DOL) 9. Systolic dysfunction was evident by ECHO in DOL1 cKO pups and progressed to frank heart failure (HF) by DOL9. Histological exam showed decreased myocardial mass with increased intercellular fibrosis. Ventricles of DOL1 cKO pups demonstrated increased expression of Acta1, Nppa, and Nppb, consistent with HF but showed decreased expression of Myh7, suggesting an impairment in the typical fetal gene program activated in HF. Myocardial immunostaining showed reduction in Ki67 (G1/S/G2/M phase marker) (Fig 1) and PH3-S10 (M phase marker) positive CMs by 82% and 46% respectively in DOL1 cKO hearts, indicating significantly reduced CM proliferation. The expression of essential cell cycle proteins showed a significant decrease in the levels of G1/S regulating proteins, CDK4, CDK6, ppRB S807/811 and S/G2 and G2/M regulating proteins, pWEE1 S642 and Cyclin B1 in cKO hearts (Fig 2). Similar results in ex vivo and in vitro Tead1 knockout models in CMs using neonatal Tead1 F/F CMs and HL1 cells validated the cell autonomous regulation of CM cell cycle by TEAD1. Conclusions: TEAD1 is required for embryonic and neonatal CM proliferation and its loss at mid gestation leads to neonatal HF associated with impaired fetal gene program activation and decreased expression of cell cycle promoting genes.

Background— Cardiomyocytes are rich in mitochondria which are situated in spatially distinct subcellular regions, including those under the plasma membrane, subsarcolemmal mitochondria, and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome–encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics. Methods and Results— Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs, that are redistributed in spatially distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RNA-induced silencing complex constituents argonaute-2 (Ago2) and fragile X mental retardation–related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the interfibrillar mitochondria following diabetic insult led to downregulation of mitochondrially encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and downregulation of functional ATP6 protein, whereas antagomir blockade restored functional ATP6 protein and cardiac pump function. Conclusions— We propose mitomiRs can translationally regulate mitochondrially encoded proteins in spatially distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RNA-induced silencing complex constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.

Aims We have previously reported alterations in cardiolipin content and inner mitochondrial membrane (IMM) proteomic make-up specifically in interfibrillar mitochondria (IFM) in the type 1 diabetic heart; however, the mechanism underlying this alteration is unknown. The goal of this study was to determine how the cardiolipin biosynthetic pathway and cardiolipin–IMM protein interactions are impacted by type 1 diabetes mellitus. Main methods Male FVB mice were made diabetic by multiple low-dose streptozotocin injections and sacrificed five weeks post-diabetic onset. Messenger RNA was measured and cardiac mitochondrial subpopulations were isolated. Further mitochondrial functional experimentation included evaluating the protein expression of the enzymes directly responsible for cardiolipin biosynthesis, as well as ATP synthase activity. Interactions between cardiolipin and ATP synthase subunits were also examined. Key findings Western blot analysis revealed a significant decrease in cardiolipin synthase (CRLS) protein content in diabetic IFM, with a concomitant decrease in its activity. ATP synthase activity was also significantly decreased. We identified two novel direct interactions between two subunits of the ATP synthase F0 complex (ATP5F1 and ATP5H), both of which were significantly decreased in diabetic IFM. Significance Overall, these results indicate that type 1 diabetes mellitus negatively impacts the cardiolipin biosynthetic pathway specifically at CRLS, contributing to decreased cardiolipin content and loss of interactions with key ATP synthase F0 complex constituents in the IFM.

Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the inner mitochondrial membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial protein import. The goal of this study was to determine whether overexpression of mitochondria phospholipid hydroperoxide glutathione peroxidase 4 (mPHGPx), an antioxidant enzyme capable of scavenging membrane-associated lipid peroxides in the IMM, could reverse proteomic alterations, dysfunctional protein import, and ultimately, mitochondrial dysfunction associated with the diabetic heart. MPHGPx transgenic mice and controls were made diabetic by multiple low-dose streptozotocin injections and examined after 5 wk of hyperglycemia. Five weeks after hyperglycemia onset, in vivo analysis of cardiac contractile function revealed decreased ejection fraction and fractional shortening in diabetic hearts that was reversed with mPHGPx overexpression. MPHGPx overexpression increased electron transport chain function while attenuating hydrogen peroxide production and lipid peroxidation in diabetic mPHGPx IFM. MPHGPx overexpression lessened proteomic loss observed in diabetic IFM. Posttranslational modifications, including oxidations and deamidations, were attenuated in diabetic IFM with mPHGPx overexpression. Mitochondrial protein import dysfunction in diabetic IFM was reversed with mPHGPx overexpression correlating with protein import constituent preservation. Ingenuity Pathway Analyses indicated that oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid oxidation processes most influenced in diabetic IFM were preserved by mPHGPx overexpression. Specific mitochondrial networks preserved included complex I and II, mitochondrial ultrastructure, and mitochondrial protein import. These results indicate that mPHGPx overexpression can preserve the mitochondrial proteome and provide cardioprotective benefits to the diabetic heart.

The observation that approximately 15% of women with disseminated breast cancer will develop symptomatic brain metastases combined with treatment guidelines discouraging single-agent chemotherapeutic strategies facilitates the desire for novel strategies aimed at outright brain metastasis prevention. Effective and robust preclinical methods to evaluate early-stage metastatic processes, brain metastases burden, and overall mean survival are lacking. Here, we develop a novel method to quantitate early metastatic events (arresting and extravasation) in addition to traditional end time-point parameters such as tumor burden and survival in an experimental mouse model of brain metastases of breast cancer. Using this method, a reduced number of viable brain-seeking metastatic cells (from 3,331 ± 263 cells/brain to 1,079 ± 495 cells/brain) were arrested in brain one week postinjection after TGFβ knockdown. Treatment with a TGFβ receptor inhibitor, galunisertib, reduced the number of arrested cells in brain to 808 ± 82 cells/brain. Furthermore, we observed a reduction in the percentage of extravasated cells (from 63% to 30%) compared with cells remaining intralumenal when TGFβ is knocked down or inhibited with galunisertib (40%). The observed reduction of extravasated metastatic cells in brain translated to smaller and fewer brain metastases and resulted in prolonged mean survival (from 36 days to 62 days). This method opens up potentially new avenues of metastases prevention research by providing critical data important to early brain metastasis of breast cancer events. Cancer Prev Res; 8(1); 68–76. ©2014 AACR.

Mitofilin, also known as heart muscle protein, is an inner mitochondrial membrane structural protein that plays a central role in maintaining cristae morphology and structure. It is a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex which is important for mitochondrial architecture and cristae morphology. Our laboratory has previously reported alterations in mitochondrial morphology and proteomic make-up during type 1 diabetes mellitus, with mitofilin being significantly down-regulated in interfibrillar mitochondria (IFM). The goal of this study was to investigate whether overexpression of mitofilin can limit mitochondrial disruption associated with the diabetic heart through restoration of mitochondrial morphology and function. A transgenic mouse line overexpressing mitofilin was generated and mice injected intraperitoneally with streptozotocin using a multi low-dose approach. Five weeks following diabetes mellitus onset, cardiac contractile function was assessed. Restoration of ejection fraction and fractional shortening was observed in mitofilin diabetic mice as compared to wild-type controls (P

The mitochondrion has been implicated in the development of diabetic cardiomyopathy. Examination of cardiac mitochondria is complicated by the existence of spatially distinct subpopulations including subsarcolemmal (SSM) and interfibrillar (IFM). Dysfunction to cardiac SSM has been reported in murine models of type 2 diabetes mellitus; however, subpopulation-based mitochondrial analyses have not been explored in type 2 diabetic human heart. The goal of this study was to determine the impact of type 2 diabetes mellitus on cardiac mitochondrial function in the human patient. Mitochondrial subpopulations from atrial appendages of patients with and without type 2 diabetes were examined. Complex I- and fatty acid-mediated mitochondrial respiration rates were decreased in diabetic SSM compared with nondiabetic ( P ≤ 0.05 for both), with no change in IFM. Electron transport chain (ETC) complexes I and IV activities were decreased in diabetic SSM compared with nondiabetic ( P ≤ 0.05 for both), with a concomitant decline in their levels ( P ≤ 0.05 for both). Regression analyses comparing comorbidities determined that diabetes mellitus was the primary factor accounting for mitochondrial dysfunction. Linear spline models examining correlative risk for mitochondrial dysfunction indicated that patients with diabetes display the same degree of state 3 and electron transport chain complex I dysfunction in SSM regardless of the extent of glycated hemoglobin (HbA1c) and hyperglycemia. Overall, the results suggest that independent of other pathologies, mitochondrial dysfunction is present in cardiac SSM of patients with type 2 diabetes and the degree of dysfunction is consistent regardless of the extent of elevated HbA1c or blood glucose levels.

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, G alpha and G beta gamma in the cytosol. In this study, we show novel nuclear functions of G beta gamma through demonstrating interaction of G beta(2) with integral components of chromatin and effects of G beta(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented G beta(2) levels in the nucleus and G beta(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of G beta(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. G beta(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the G beta(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of G beta gamma with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific "lid" conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar(1), Gln(2), Ile(8)] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.

Dysfunctional mitochondria are central in the pathogenesis of diabetic cardiomyopathy. Mitochondrial proteomic alterations resulting from diabetes mellitus have been reported although the mechanisms driving changes in proteomic signatures are unknown. microRNAs (miRNAs) have been considered as potential regulators of proteins. The goal of this study was to determine whether miRNAs play a role in diabetes-induced mitochondrial proteomic alterations. Quanitative RT-PCR miRNA screening in diabetic mice, 5 wk following multiple low-dose streptozotocin treatment was associated with alteration in the expression of 29 miRNAs in the diabetic heart compared with control. Among those miRNAs upregulated in the diabetic heart was miR-141 ( P < 0.002). miRNA target prediction analyses identified miR-141 as a potential regulator of the inner mitochondrial membrane phosphate transporter, solute carrier family 25 member 3 (Slc25a3), which provides inorganic phosphate to the mitochondrial matrix and is essential for ATP production. With the use of a luciferase reporter construct with a Slc25a3 3′-untranslated region (UTR) target sequence, overexpression of miR-141 downregulated luciferase activity levels confirming miR-141/Slc25a3 3′-UTR binding. miR-141 overexpression in HL-1 cells elicited a decrease in Slc25a3 protein content, ATP production and a decrease in ATP synthase activity, similar to the diabetic phenotype ( P < 0.05, for both). Diabetic interfibrillar mitochondria (IFM) displayed decreased Slc25a3 protein content, which was inversely correlated with increased miR-141 expression. Further, diabetic IFM ATP synthase activity was also decreased ( P < 0.05). Together these results indicate that miR-141 can regulate Slc25a3 protein expression in the diabetic heart. Further, diabetes-induced miRNA changes may influence mitochondrial proteomes and functional processes such as mitochondrial ATP production.

Receptor-activating autoantibodies targeting different G-protein-coupled receptors (GPCRs) have been discovered that exhibit agonist-like activity in several human pathologies. For example, autoimmune pathogenesis of Graves’ disease is attributed to autoantibody-mediated activation of the thyrotropin receptor, a GPCR. Likewise, diseases such as preeclampsia and vascular allograft rejection are caused by autoantibodies against angiotensin II type 1 receptor (AT1R). The serum of patients with Chagas disease causing congestive heart failure contains an autoantibody for the β1-adrenergic receptor. Autoantibodies against α1- and β1- and β2-adrenergic receptors found in serum from patients are linked to malignant hypertension and idiopathic dilated cardiomyopathy, respectively. Additional examples of GPCR-activating antibodies include those against the mGluR, GABA, 5HT4, calcium-sensing receptor, muscarinic M1 and M2 receptors, which have been identified in various chronic neurological diseases patients. The GPCR-directed autoantibodies may actually initiate the cellular signaling responsible for the disease since each disorder is associated with a specific GPCR-directed autoantibody. Empirical evidence suggests that the autoantibody induces GPCR activation without the endogenous ligand; however, the mechanism of antibody mediated receptor activation is not known. We show that the conformational dynamics of the extracellular domain of the AT1R generates the epitope for an autoantibody on the plasma membrane surface. This allows the antibody to bind and stabilize the activated state of AT1R, thus providing a molecular basis for the autoantibody action.

Chronic activation of angiotensin II (AngII) type 1 receptor (AT(1)R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT(1)R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT(1)R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT(1)R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT(1)R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT(1)R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.

The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.

Diaminopropionate (DAP) ammonia lyase (a PLP-dependent enzyme; EC 4.3.1.15) catalyzes the \alpha,\beta-elimination reaction of both L- and D-\alpha,\beta-diaminopropionate to form pyruvate and ammonia. Escherichia coli DAP ammonia lyase gene was cloned and overexpressed in E. coli and the protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. Crystals of two different morphologies were obtained, one of which belonged to the tetragonal space group $P4_12_12$ (or $P4_32_12$), with unit-cell parameters a = b = 86.01, c = 209.56 \AA, and the other to the monoclinic space group $P_21$, with unit-cell parameters a = 87.78, b = 94.35, c = 96.02 \AA, = 109.73°. The tetragonal crystals diffracted X-rays to 3.0 \AA resolution, while diffraction from the monoclinic form extended to 2.5 \AA. Complete X-ray diffraction data sets have been collected for both crystal forms.