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Signals generated by cryptochrome (CRY) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. The CRYs are also identified as circadian blue-light photoreceptors in Drosophila and components of the mammalian circadian clock. These flavoproteins all have an N-terminal domain that is similar to photolyase, and most have an additional C-terminal domain of variable length. We present here the crystal structure of the photolyase-like domain of CRY-1 from Arabidopsis thaliana . The structure reveals a fold that is very similar to photolyase, with a single molecule of FAD noncovalently bound to the protein. The surface features of the protein and the dissimilarity of a surface cavity to that of photolyase account for its lack of DNA-repair activity. Previous in vitro experiments established that the photolyase-like domain of CRY-1 can bind Mg·ATP, and we observe a single molecule of an ATP analog bound in the aforementioned surface cavity, near the bound FAD cofactor. The structure has implications for the signaling mechanism of CRY blue-light photoreceptors.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the formation of mevalonate, the committed step in the biosynthesis of sterols and isoprenoids. The activity of HMGR is controlled through synthesis, degradation and phosphorylation to maintain the concentration of mevalonate-derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels. Three crystal structures of the catalytic portion of human HMGR in complexes with HMG-CoA, with HMG and CoA, and with HMG, CoA and NADP(+), provide a detailed view of the enzyme active site. Catalytic portions of human HMGR form tight tetramers. The crystal structure explains the influence of the enzyme's oligomeric state on the activity and suggests a mechanism for cholesterol sensing. The active site architecture of human HMGR is different from that of bacterial HMGR; this may explain why binding of HMGR inhibitors to bacterial HMGRs has not been reported.

Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution. Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport. The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Glioblastoma Multiforme (GBM) is the most frequent of malignant tumors of the brain. It is fatal usually within fifteen months after diagnosis. The Cancer Genome Atlas (TCGA) has surveyed the genomes of hundreds of human GBMs for presence of mutations in genes previously associated with cancer. Among those identified, three of the five most frequently mutated genes encode the well-known tumor suppressors: P53, NF1, and Pten (TCGA, 2008). We have generated genetic mouse models of GBM based on mutation in these same three tumor suppressors and they develop GBM with 100% incidence. These mouse GBMs resemble the human counterparts by all commonly used criteria for diagnosis. Extensive study of these mice and tumors has provided novel insights into the natural history of GBM development and moreover pointed at the adult neural stem cell/progenitor compartment as the most likely and frequent source of these tumors (Kwon CH, et al., 2008). We have thus isolated a cancer stem cell and demonstrated a hierarchical growth for these tumors in vivo. (Chen J, et al., 2012) In an effort to identify novel GBM specific molecular pathways that might serve as therapeutic targets, we undertook a 200,000 small compound high throughput screen using primary low passage GBM cells generated by pooling cells from multiple mouse tumors (Mut6 cells). A critical counter screen was designed to minimize compounds that targeted the cell division and DNA replication machinery of cells since many normal cells divide and this property is not unique to cancer cells. We therefore ruled out all compounds that displayed toxicity to primary mouse embryo fibroblasts (MEFs) or to primary astrocytes. At the end of the screen, we selected three lead compounds with amenable physical-chemical properties for further evaluation and development as research discovery reagents and possible translational tools for drug development. We verified the activity of one of these compounds, termed Compound X, on mouse and human GBM primary cells as well as on a subset of established cancer cell lines, but not on MEFs or primary astrocytes. We identified a rapid cellular stress response accompanied by the robust activation of a transcription factor and eventual apoptotic response that can be mimicked by glucose deprivation. Through intensive structure-activity relationship study, analogs were successfully developed for in vivo activity and also for biochemical pull-down assay. Another compound (Compound Y) also induced apoptosis, however displaying response through completely different pathway, which indicates our screen identified multiple vulnerabilities in GBM tumor cells. Citation Format: Sang Kyun Lim, Yufeng Shi, Qiren Liang, Inga Nazarenko, Shuguang Wei, Maya Palnitkar, Yanjiao Li, Woosung Cho, Noelle Williams, Bruce Posner, Jef De Brabander, Luis F. Parada. Identification of small molecules for GBM study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A63.

Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of Thermus thermophilus UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process.

The hyt/hyt mouse has a severe and pervasive primary inherited hypothyroidism with significantly depressed serum T4, elevated serum and pituitary TSH, and reduced thyroid gland iodide uptake. Previous ultrastructural and histologic analysis of the hyt/hyt thyroid gland along with these biochemical abnormalities support an inherited defect in TSH responsiveness of the hyt/hyt thyroid gland. In order to evaluate the potential site of the defect in the hyt/hyt mouse, we have studied the hyt/hyt gland and hyt/hyt TSH from a biochemical and molecular standpoint. Based on demonstrated bioactivity of hyt/hyt serum in the McKenzie bioassay, this reduced responsiveness to TSH in the hyt/hyt mouse is not due to reduced bioactivity of hyt/hyt TSH or a major structural abnormality in the hyt/hyt TSH molecule. In comparison to hyt/ + euthyroid littermates and +/+ BALB/cBY progenitor strain mice, the hyt/hyt mouse demonstrates a twofold reduction in thyroid gland basal cAMP and a markedly diminished response of adenylyl cyclase to exogenous TSH. However, hyt/hyt cAMP production is equivalent to the euthyroid mice after stimulation of thyroid glands by forskolin, cholera toxin, PGE1, and isoproterenol. These results support a defect in the TSH-G protein-adenylyl cyclase system in the hyt/hyt thyroid gland. Specifically, these findings suggest that the hyt/hyt mouse has a defect in TSH responsivity due to an inherited defect in the thyroid gland TSH receptor molecule. Since the hyt/hyt gland makes T3 and T4 but at diminished levels, the proposed defect in the TSH receptor would still impart partial function. Both hyt/hyt and euthyroid hyt/ + littermates make TSH receptor mRNAs of 5500 and 2400 base pairs. This suggests that the receptor defect does not represent a major structural abnormality of the gene. The receptor defect could represent a reduction in receptor number, receptor-TSH affinity, or TSH receptor-G protein coupling. The specificity of this effect on adenylyl cyclase-cAMP is shown by the reduction of TSH-cAMP regulated thyroid peroxidase (TPO) and thyroglobulin mRNAs in the hyt/hyt thyroid gland. Given the importance of TPO and thyroglobulin in normal thyroid hormone synthesis, the reductions in TPO and thyroglobulin mRNAs in the hyt/hyt thyroid gland may underlie the significant decrease in thyroid hormone production by the hyt/hyt mouse.

Thyroid hormones, T3 and T4, have been shown to play significant but poorly understood roles in development and differentiation of rodent and human brain(Lauder, 1989; Legrand, 1982–83; Stein et al, 1989a; 1991a,d; Eayrs, 1968; Morreale de Escobar et al, 1984; Garza et al, 1988; Ruiz-Marcos, 1989; Nunez et al, 1989). Hypothyroidism leads to molecular(Stein et al, 1989a,c; 1991a; Nunez et al, 1989; Hendrich et al, 1987), neuroendocrinological(Noguchi et al, 1986, Bakke et al, 1975, Stein et al, 1989b, Porterfield et al, 1981), neuroanatomical(Lauder et al, 1986; Lauder, 1989; Ruiz-Marcos, 1989; Eayrs, 1955; Garza et al, 1988; Morreale de Escobar et al, 1989; Marc et al, 1985; Legrand, 1982–83; Rami et al, 1986b; Narayanan et al, 1985; Marinesco, 1924; Lotmar, 1928; Rosman, 1975), behavioral and neuropsychological(Adams et al, 1989,1991; Anthony et al, 1991; Eayrs, 1968; Davenport et al, 1976; Klein, 1985; Rovet et al, 1987; Rovet, 1989; Man, 1971; Boyages et al, 1988; Pharoah,1984), and neurological abnormalities(Chaouki et al, 1989; Boyages et al, 1988; Delong et al, 1985; Nelson et al, 1986; Macfaul et al; 1978; Stein et al, 1991d, Rochiccioli et al, 1989) in the developing brain. Specifically, disorders of neuronal process growth and connectivity are noted neuroanatomically and motor syndromes involving motor cortex and pyramidal tracts are commonly observed in hypothyroid humans and rodents. These neurological and neuropathological abnormalities may be predicated on abnormalities in the cytoskeletal structures and in their molecular components. The cytoskeleton is a primary target for thyroid hormone in euthyroid and hypothyroid brain.

Mice homozygous for the autosomal-recessive gene hypothyroid (hyt) had congenital hypothyroidism of fetal onset after 15 days postconception. Neonatal hyt/hyt mice had reduced serum thyroxine ranging from 1/5 to 1/6 of normal as well as significantly delayed somatic and behavioral development. Delayed somatic development included retarded eye opening and ear raising, and reduced body length and body weight. The hyt/hyt animals compared to their normal littermates demonstrated delayed reflexive behavior and abnormal motor and adaptive behavior. The somatic and behavioral measures clearly distinguished hyt/hyt animals from their normal littermates even without T4 determination. The somatic and reflexive behavioral abnormalities in the hyt/hyt mouse were similar to other rodent models of human congenital hypothyroidism. The hyt/hyt mouse provided an ideal model for exploring the effect of severe primary inherited hypothyroidism related to deficient autonomous fetal thyroid function and was consistent with the hypothesis that thyroid hormone deficit in utero and in the early neonatal period significantly altered functional development.

Thyroid hormones, T3 and T4, have been shown to play significant but poorly understood roles in development and differentiation of rodent and human brain (1–7). In the human, disorders of maternal and fetal thyroid function include maternal and secondary fetal iodine deficiency, maternal hypothyroidism or hyperthyroidism, as well as disorders related to deficient fetal autonomous thyroid hormone secretion, i.e., goiter or sporadic congenital hypothyroidism. These disorders are identifiable causes of mental retardation (4, 8, 9, 10), cerebral palsy (11, 12), and other significant neurological abnormalities (5, 6, 11).