Zheng Zheng Shi

Coordination chemistry based approach to lipophilic inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate pathway found in most bacteria is a validated anti-infective drug target. Fosmidomycin, a potent DXR inhibitor, is active against Gram-negative bacteria. A coordination chemistry and structure based approach was used to discover a novel, lipophilic DXR inhibitor with an IC(50) of 1.4 microM. It exhibited a broad spectrum of activity against Gram-negative and -positive bacteria with minimal inhibition concentrations of 20-100 microM (or 3.7-19 microg/mL).

OLA1, an Obg-like ATPase, suppresses antioxidant response via nontranscriptional mechanisms

Oxidative stress has been implicated in diverse disease states and aging. To date, induction of cellular responses to combat oxidative stress has been characterized largely at the transcriptional level, with emphasis on Nrf2-mediated activation of antioxidant response elements. In this study, we demonstrate that OLA1, a novel Obg-like ATPase, functions as a negative regulator of the cellular antioxidant response independent of transcriptional processes. Knockdown of OLA1 in human cells elicited an increased resistance to oxidizing agents including tert-butyl hydroperoxide (tBH) and diamide without affecting cell proliferation, baseline apoptosis, or sensitivity to other cytotoxic agents that target the mitochondria, cytoskeleton, or DNA. Conversely, overexpression of OLA1 increased cellular sensitivity to tBH and diamide. When challenged with oxidants, OLA1-knockdown cells had decreased production of intracellular reactive oxygen species and exhibited less depletion of reduced glutathione. Surprisingly, knockdown of OLA1 caused only minimal genomic response; no changes were found in the mRNA levels of genes encoding antioxidant enzymes, enzymes that produce antioxidants (including glutathione), or other genes known to respond to Nrf2. Moreover, when de novo protein synthesis was blocked by cycloheximide in OLA1-knockdown cells, they continued to demonstrate increased resistance to both tBH and diamide. These data demonstrate that OLA1 suppresses the antioxidant response through nontranscriptional mechanisms. The beneficial effects observed upon OLA1-knockdown suggest that this regulatory ATPase is a potential novel target for antioxidative therapy.

Identification of two additional members of the membrane-bound dipeptidase family

We have cloned two mouse cDNAs encoding previously unidentified membrane‐bound dipeptidases [membrane‐bound dipeptidase‐2 (MBD‐2) and membrane‐bound dipeptidase‐3 (MBD‐3)] from membrane‐bound dipeptidase‐1 (MBD‐1) deficient mice (Habib, G.M., Shi, Z‐Z., Cuevas, A.A., Guo, Q., Matzuk, M.M., and Lieberman, M.W. (1998) Proc. Natl. Acad. Sci. USA 95, 4859–4863). These enzymes are closely related to MBD‐1 (EC 3.4.13.19), which is known to cleave leukotriene D4 (LTD4) and cystinyl‐bis‐glycine. MBD‐2 cDNA is 56% identical to MBD‐1 with a predicted amino acid identity of 33%. The MBD‐3 and MBD‐1 cDNAs share a 55% nucleotide identity and a 39% predicted amino acid sequence identity. All three genes are tightly linked on the same chromosome. Expression of MBD‐2 and MBD‐3 in Cos cells indicated that both are membrane‐bound through a glycosylphosphatidyl‐inositol linkage. MBD‐2 cleaves leukotriene D4 (LTD4) but not cystinyl‐bis‐glycine, while MBD‐3 cleaves cystinyl‐bis‐glycine but not LTD4. MBD‐1 is expressed at highest levels in kidney, lung, and heart and is absent in spleen, while MBD‐2 is expressed at highest levels in lung, heart, and testis and at somewhat lower levels in spleen. Of the tissues examined, MBD‐3 expression was detected only in testis. Our identification of a second enzyme capable of cleaving LTD4 raises the possibility that clearance of LTD4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.

Glutathione is essential for early embryogenesis – Analysis of a glutathione synthetase knockout mouse

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene (Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.

OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation

Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression.

Regulation of cell-matrix adhesion by OLA1, the Obg-like ATPase 1

Attachment of cells to the extracellular matrix induces clustering of membrane receptor integrins which in turn triggers the formation of focal adhesions (FAs). The adaptor/scaffold proteins in FAs provide linkage to actin cytoskeleton, whereas focal adhesion kinase (FAK) and other FA-associated kinases and phosphatases transduce integrin-mediated signaling cascades, promoting actin polymerization and progression of cell spreading. In this study, we explored the role of OLA1, a newly identified member of Obg-like ATPases, in regulating cell adhesion processes. We showed that in multiple human cell lines RNAi-mediated downregulation of OLA1 significantly accelerated cell adhesion and spreading, and conversely overexpression of OLA1 by gene transfection resulted in delayed cell adhesion and spreading. We further found that OLA1-deficient cells had elevated levels of FAK protein and decreased Ser3 phosphorylation of cofilin, an actin-binding protein and key regulator of actin filament dynamics, while OLA1-overexpressing cells exhibited the opposite molecular alterations in FAK and cofilin. These findings suggest that OLA1 plays an important negative role in cell adhesion and spreading, in part through the regulation of FAK expression and cofilin phosphorylation, and manipulation of OLA1 may lead to significant changes in cell adhesion and the associated phenotypes.

OLA1 contributes to epithelial-mesenchymal transition in lung cancer by modulating the GSK3β/snail/E-cadherin signaling

Obg-like ATPase 1 (OLA1) belongs to the Obg family of P-loop NTPases, and may serve as a “molecular switch” regulating multiple cellular processes. Aberrant expression of OLA1 has been observed in several human malignancies. However, the role of OLA1 in cancer progression remains poorly understood. In this study, we used the Kaplan-Meier plotter search tool to show that increased expression of OLA1 mRNA was significantly associated with shorter overall survival in lung cancer patients. By immunohistochemical analysis we discovered that levels of OLA1 protein in lung cancer tissues were positively correlated with TNM stage and lymph node metastasis, but negatively correlated with the epithelial-mesenchymal transition (EMT) marker E-cadherin. Knockdown of OLA1 in a lung adenocarcinoma cell line rendered the cells more resistant to TGF- β-induced EMT and the accompanied repression of E-cadherin. Furthermore, our results demonstrated that OLA1 is a GSK3 β-interacting protein and inhibits GSK3 β activity by mediating its Ser9 phosphorylation. During EMT, OLA1 plays an important role in suppressing the GSK3 β-mediated degradation of Snail protein, which in turn promotes downregulation of E-cadherin. These data suggest that OLA1 contributes to EMT by modulating the GSK3 β/Snail/E-cadherin signaling, and its overexpression is associated with clinical progression and poor survival in lung cancer patients.

Obg-like ATPase 1 regulates global protein serine/threonine phosphorylation in cancer cells by suppressing the GSK3β- inhibitor 2-PP1 positive feedback loop

OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif.

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound

Pulsed high intensity focused ultrasound (HIFU) produced has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure >7 MPa (∼27 W acoustic power at 1.4 MHz) destroys >95 % of breast cancer cells MDA-MB-231 in suspension with >10 μM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (Nacetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected ...