Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn341, Tyr60 and Phe351 in tetrahydrofolate binding

2009 ◽  
Vol 418 (3) ◽  
pp. 635-642 ◽  
Author(s):  
Vinitha R. Pai ◽  
V. Rajaram ◽  
Shveta Bisht ◽  
B. S. Bhavani ◽  
N. Appaji Rao ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Bacillus Stearothermophilus ◽  
Side Chain ◽  
Side Reactions ◽  
Type I ◽  
Functional Studies ◽  
Binary Complexes ◽  
Dependent Activity ◽  
In The Wild

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5′-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of β-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn341, Tyr60 and Phe351, which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe351 to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn341 and Tyr60 are pivotal for the binding of THF/FTHF, whereas Phe351 is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Design and Crystal Structures of Triple Helicates with Crystallographic IdealizedD3Symmetry:  The Role of Side Chain Effect on Crystal Packing

2002 ◽  
Vol 41 (23) ◽  
pp. 5978-5985 ◽  
Author(s):  
Guo Dong ◽  
Pang Ke-liang ◽  
Duan Chun-ying ◽  
He Cheng ◽  
Meng Qing-jin
Keyword(s):  
Crystal Structures ◽  
Crystal Packing ◽  
Side Chain

scholarly journals Crystal structures of GCN2 C-terminal domain: Insight into GCN2 regulation

2014 ◽  
Vol 70 (a1) ◽  
pp. C1407-C1407
Author(s):  
Isha Singh ◽  
Hongzhen He ◽  
Sheree Wek ◽  
Souvik Dey ◽  
Thomas Baird ◽  
...  
Keyword(s):  
Amino Acid ◽  
Crystal Structures ◽  
Kinase Domain ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Nucleic Acid Binding ◽  
Serine Threonine Kinase ◽  
Functional Studies ◽  
Terminal Domain

General control non-derepressible 2 kinase (GCN2) is a serine threonine kinase that curtails translation in response to diverse stress stimuli [1]. It is a primary sensor of amino acid starvation and mediates translation repression by phosphorylating eIF2 [2]. In addition to the kinase domain, GCN2 contains two regulatory regions; a histidyl-tRNA synthetase-like domain (HisRS) and a C-terminal domain (CTD), which function together to sense nutrient depletion. Both domains have been proposed to bind uncharged tRNA's that accumulate during amino acid starvation followed by dimerization of the kinase domain facilitating activation of GCN2 [3]. Thus, while the CTD plays an important regulatory role in activating GCN2, information on how the CTD facilitates dimerization and whether the CTD plays a similar role in murine GCN2 is limited. Moreover, the sequences of vertebrate CTDs share less than 10% sequence identity with their yeast counterpart; therefore, it is not known whether regulatory mechanisms in GCN2 are conserved across different species. We present here the experimentally phased crystal structures of murine CTD at 1.9 Å and yeast CTD at 1.95 Å. Both murine and yeast CTDs share a novel interdigitated dimeric organization, although the dimeric structures differ somewhat in overall shape and size. Additional biochemical analysis of the murine CTD confirms an important role for dimerization in its activation. Moreover, functional studies reveal that both yeast and murine GCN2 have similar nucleic acid binding properties, but mGCN2 does not appear to exhibit ribosomal association, a key feature in the model for regulation of yeast GCN2, suggesting that there are regulatory differences between the murine GCN2 and its yeast counterpart. Our data provides a basis for understanding the role of the CTD in regulation of GCN2 in both yeast and mammals.

scholarly journals Role of Transferrin Receptor from a Neisseria meningitidis tbpB Isotype II Strain in Human Transferrin Binding and Virulence

2004 ◽  
Vol 72 (6) ◽  
pp. 3461-3470 ◽  
Author(s):  
Geneviève Renauld-Mongénie ◽  
David Poncet ◽  
Michèle Mignon ◽  
Sophie Fraysse ◽  
Christophe Chabanel ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Transferrin Receptor ◽  
Iron Uptake ◽  
Bacterial Virulence ◽  
Type I ◽  
Wild Type ◽  
Antigenic Properties ◽  
In The Wild ◽  
Isogenic Mutants

ABSTRACT Neisseria meningitidis acquires iron through the action of the transferrin (Tf) receptor, which is composed of the Tf-binding proteins A and B (TbpA and TbpB). Meningococci can be classified into isotype I and II strains depending on whether they harbor a type I or II form of TbpB. Both types of TbpB have been shown to differ in their genomic, biochemical, and antigenic properties. Here we present a comparative study of isogenic mutants deficient in either or both Tbps from the isotype I strain B16B6 and isotype II strain M982. We show that TbpA is essential in both strains for iron uptake and growth with iron-loaded human Tf as a sole iron source. No growth has also been observed for the TbpB− mutant of strain B16B6, as shown previously, whereas the growth of the analogous mutant in M982 was similar to that in the wild type. This indicates that TbpB in the latter strain plays a facilitating but not essential role in iron uptake, which has been observed previously in similar studies of other bacteria. These data are discussed in relation to the fact that isotype II strains represent more than 80% of serogroup B meningococcal strains. The contribution of both subunits in the bacterial virulence of strain M982 has been assessed in a murine model of bacteremia. Both the TbpB− TbpA− mutant and the TbpA− mutant are shown to be nonvirulent in mice, whereas the virulence of the TbpB− mutant is similar to that of the wild type.

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

Isoform-Selective PI3K Inhibitors for Various Diseases

2020 ◽  
Vol 20 (12) ◽  
pp. 1074-1092 ◽  
Author(s):  
Rammohan R.Y. Bheemanaboina
Keyword(s):  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Type I ◽  
Selective Inhibitors ◽  
Pi3k Inhibitors ◽  
Wide Range ◽  
Lipid Kinases ◽  
Isoform Selectivity

Phosphoinositide 3-kinases (PI3Ks) are a family of ubiquitously distributed lipid kinases that control a wide variety of intracellular signaling pathways. Over the years, PI3K has emerged as an attractive target for the development of novel pharmaceuticals to treat cancer and various other diseases. In the last five years, four of the PI3K inhibitors viz. Idelalisib, Copanlisib, Duvelisib, and Alpelisib were approved by the FDA for the treatment of different types of cancer and several other PI3K inhibitors are currently under active clinical development. So far clinical candidates are non-selective kinase inhibitors with various off-target liabilities due to cross-reactivities. Hence, there is a need for the discovery of isoform-selective inhibitors with improved efficacy and fewer side-effects. The development of isoform-selective inhibitors is essential to reveal the unique functions of each isoform and its corresponding therapeutic potential. Although the clinical effect and relative benefit of pan and isoformselective inhibition will ultimately be determined, with the development of drug resistance and the demand for next-generation inhibitors, it will continue to be of great significance to understand the potential mechanism of isoform-selectivity. Because of the important role of type I PI3K family members in various pathophysiological processes, isoform-selective PI3K inhibitors may ultimately have considerable efficacy in a wide range of human diseases. This review summarizes the progress of isoformselective PI3K inhibitors in preclinical and early clinical studies for anticancer and other various diseases.

The Role of Toll-Like Receptors and Type I Interferons in Host Responses to Bacteria

2009 ◽  
Vol 5 (2) ◽  
pp. 143-149
Author(s):  
Marja Ojaniemi ◽  
Mari Liljeroos ◽  
Reetta Vuolteenaho
Keyword(s):  
Type I Interferons ◽  
Host Responses ◽  
Type I ◽  
Toll Like Receptors

scholarly journals Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor

2021 ◽  
Vol 13 (1) ◽  
pp. 17-29
Author(s):  
Emann M Rabie ◽  
Sherry X Zhang ◽  
Andreas P Kourouklis ◽  
A Nihan Kilinc ◽  
Allison K Simi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Type I ◽  
Matrix Degradation ◽  
Human Breast ◽  
Rate Of Invasion

Abstract Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

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