The structure and mechanism of SAICAR synthetase

SAICAR synthetase catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis: ATP + L-aspartate + CAIR → ADP + Pi + SAICAR Enzymes involved in nucleotide synthesis are common targets for chemotherapeutic and antimicrobial drugs. They work by reducing the rate of growth of rapidly proliferating cells, thus preventing tumor growth and microbial fecundity. L-Alanosine, an L-aspartate analogue that acts as a substrate for SAICAR synthetase, is a natural product with antiviral and antitumor activities. The product of the reaction is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase and is responsible for L-alanosine toxicity. L-Alanosine may be effective as a chemotherapeutic agent in combination with other drugs for certain T-Cell lymphocyte tumors.;Crystal structures of E. coli SAICAR synthetase with CAIR revealed two new Mg2+ binding sites. The binding of CAIR and metals orders a loop (residues 35-39). Site-directed mutagenesis experiments and crystal structures reveal a role in L-aspartate binding for this loop. Analogs of CAIR (AICAR and IMP) are phosphorylated, as revealed by crystal structures, and likely mimic a putative phosphoryl intermediate of the substrate. Positional Isotope Exchange (PIX) experiments show migration of 18O from the bridging position of gamma-18O-ATP to terminal positions of the beta-phosphoryl group only in the presence of CAIR, indicative of the formation of a phosphoryl intermediate.;The enzyme from humans combines SAICAR synthetase and AIR carboxylase activities. The kinetic mechanism of human SAICAR synthetase was Steady State Ordered (CAIR first, ATP second, and L-aspartate last). Elimination of AIR carboxylase activity by mutation or by direct inhibition causes a 10-fold difference in the on rate constant for CAIR, suggesting a functional linkage between the AIR carboxylase and SAICAR synthetase active sites

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively

Sequential multi-locus transcranial magnetic stimulation for treatment of obsessive-compulsive disorder with comorbid major depression: A case series

Obsessive-compulsive disorder (OCD) and major depressive disorder (MDD) are highly comorbid [1], with depressive symptoms amplifying the chronicity and severity of OCD symptoms. Comorbid illness decreases quality of life and daily functioning [2] and is associated with greater suicidality and more frequent inpatient hospitalizations [3]. Furthermore, comorbid OCD/depression is associated with poorer response to OCD-focused psychological and pharmacological treatments [4]. Epidemiologic studies have shown that OCD symptoms generally precedes the occurrence of depression, suggesting a causal interacting model in which OCD predisposes to development of depressive symptoms [5]. In line with that causal model, Tadayonnejad et al. showed aberrant effective (directional) connectivity between OCD and MDD circuits may be a potential network mechanism of depressive symptom genesis or worsening in OCD-MDD [6]. The challenging nature of this comorbidity necessitates the development of novel, more effective treatments

Sequential multi-locus transcranial magnetic stimulation for treatment of obsessive-compulsive disorder with comorbid major depression: A case series

Obsessive-compulsive disorder (OCD) and major depressive disorder (MDD) are highly comorbid [1], with depressive symptoms amplifying the chronicity and severity of OCD symptoms. Comorbid illness decreases quality of life and daily functioning [2] and is associated with greater suicidality and more frequent inpatient hospitalizations [3]. Furthermore, comorbid OCD/depression is associated with poorer response to OCD-focused psychological and pharmacological treatments [4]. Epidemiologic studies have shown that OCD symptoms generally precedes the occurrence of depression, suggesting a causal interacting model in which OCD predisposes to development of depressive symptoms [5]. In line with that causal model, Tadayonnejad et al. showed aberrant effective (directional) connectivity between OCD and MDD circuits may be a potential network mechanism of depressive symptom genesis or worsening in OCD-MDD [6]. The challenging nature of this comorbidity necessitates the development of novel, more effective treatments

Subthreshold stimulation intensity is associated with greater clinical efficacy of intermittent theta-burst stimulation priming for Major Depressive Disorder

Background: Intermittent theta-burst stimulation priming (iTBS-P) can improve clinical outcome of patients with Major Depressive Disorder (MDD) who do not show early benefit from 10 Hz stimulation of left dorsolateral prefrontal cortex (DLPFC), also known as high-frequency left-sided (HFL) stimulation. The intensity and pulse number for iTBS-P needed to induce clinical benefit have not been systematically examined. Objective: To study the effect of intensity and pulse number on the clinical efficacy of iTBS-P. Methods: We conducted a retrospective review of 71 participants who received at least five sessions of HFL with limited clinical benefit and received iTBS-P augmentation for between 5 and 25 sessions. Intensity of iTBS-P priming stimuli ranged from 75 to 120% of motor threshold (MT) and pulse number ranged from 600 to 1800. Associations among intensity, pulse number, and clinical outcome were analyzed using a mixed methods linear model with change in IDS-SR as the primary outcome variable, priming stimulation intensity (subthreshold or suprathreshold), pulse number (1200 pulses), and gender as fixed factors, and number of iTBS-P treatments and age as continuous covariates. Results: Subjects who received subthreshold intensity iTBS-P experienced greater reduction in depressive symptoms than those who received suprathreshold iTBS-P (p = 0.011) with no effect of pulse number after controlling for stimulus intensity. Conclusions: Subthreshold intensity iTBS-P was associated with greater clinical improvement than suprathreshold stimulation. This finding is consistent with iTBS-P acting through homeostatic plasticity mechanisms

The structure and mechanism of SAICAR synthetase

SAICAR synthetase catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis: ATP + L-aspartate + CAIR → ADP + Pi + SAICAR Enzymes involved in nucleotide synthesis are common targets for chemotherapeutic and antimicrobial drugs. They work by reducing the rate of growth of rapidly proliferating cells, thus preventing tumor growth and microbial fecundity. L-Alanosine, an L-aspartate analogue that acts as a substrate for SAICAR synthetase, is a natural product with antiviral and antitumor activities. The product of the reaction is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase and is responsible for L-alanosine toxicity. L-Alanosine may be effective as a chemotherapeutic agent in combination with other drugs for certain T-Cell lymphocyte tumors.;Crystal structures of E. coli SAICAR synthetase with CAIR revealed two new Mg2+ binding sites. The binding of CAIR and metals orders a loop (residues 35-39). Site-directed mutagenesis experiments and crystal structures reveal a role in L-aspartate binding for this loop. Analogs of CAIR (AICAR and IMP) are phosphorylated, as revealed by crystal structures, and likely mimic a putative phosphoryl intermediate of the substrate. Positional Isotope Exchange (PIX) experiments show migration of 18O from the bridging position of gamma-18O-ATP to terminal positions of the beta-phosphoryl group only in the presence of CAIR, indicative of the formation of a phosphoryl intermediate.;The enzyme from humans combines SAICAR synthetase and AIR carboxylase activities. The kinetic mechanism of human SAICAR synthetase was Steady State Ordered (CAIR first, ATP second, and L-aspartate last). Elimination of AIR carboxylase activity by mutation or by direct inhibition causes a 10-fold difference in the "on" rate constant for CAIR, suggesting a functional linkage between the AIR carboxylase and SAICAR synthetase active sites.</p

Cannabis effects on brain structure, function, and cognition: considerations for medical uses of cannabis and its derivatives.

Background: Cannabis is the most widely used illicit substance worldwide, and legalization for recreational and medical purposes has substantially increased its availability and use in the United States.Objectives: Decades of research have suggested that recreational cannabis use confers risk for cognitive impairment across various domains, and structural and functional differences in the brain have been linked to early and heavy cannabis use.Methods: With substantial evidence for the role of the endocannabinoid system in neural development and understanding that brain development continues into early adulthood, the rising use of cannabis in adolescents and young adults raises major concerns. Yet some formulations of cannabinoid compounds are FDA-approved for medical uses, including applications in children.Results: Potential effects on the trajectory of brain morphology and cognition, therefore, should be considered. The goal of this review is to update and consolidate relevant findings in order to inform attitudes and public policy regarding the recreational and medical use of cannabis and cannabinoid compounds.Conclusions: The findings point to considerations for age limits and guidelines for use

Purification, Crystallization and Preliminary Crystallographic Analysis of the SH2 Domain of IL-2-inducible T-cell Kinase

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X–Pro imide bond readily undergoes cis–trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis–trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis–trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.This article is from Acta Crystallographica Section F 67 (2011): 269, doi:10.1107/S1744309110052346.</p

Purification, Crystallization and Preliminary Crystallographic Analysis of the SH2 Domain of IL-2-inducible T-cell Kinase

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X–Pro imide bond readily undergoes cis–trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis–trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis–trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.This article is from Acta Crystallographica Section F 67 (2011): 269, doi:10.1107/S1744309110052346.</p

Structure of the Interleukin-2 Tyrosine Kinase Src Homology 2 Domain; Comparison Between X-ray and NMR-Derived Structures

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis–trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis–trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively.This article is from Acta Crystallographica Section F 68 (2012): 145, doi:10.1107/S1744309111049761.</p