Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone ¹⁵N spin relaxation and ¹⁵N,¹H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.     

Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu

Mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic precursor cells predominantly found in the bone marrow. They have been recently reported to home towards the hypoxic tumor microenvironment in vivo. Interleukin-6 is a multifunctional cytokine normally involved in the regulation of the immune and inflammatory response. In addition to its normal function, IL-6 signaling has been implicated in tumorigenesis. Solid tumors develop hypoxia as a result of inadequate O₂ supply. Interestingly, tumor types with increased levels of hypoxia are known to have increased resistance to chemotherapy as well as increased metastatic potential. Here, we present evidence that under hypoxic conditions (1.5% O₂) breast cancer cells secrete high levels of IL-6, which serve to activate and attract MSCs. We now report that secreted IL-6 acts in a paracrine fashion on MSCs stimulating the activation of both Stat3 and MAPK signaling pathways to enhance migratory potential and cell survival. Inhibition of IL-6 signaling utilizing neutralizing antibodies leads to attenuation of MSC migration. Specifically, increased migration is dependent on IL-6 signaling through the IL-6 receptor. Collectively, our data demonstrate that hypoxic tumor cells specifically recruit MSCs, which through activation of signaling and survival pathways facilitate tumor progression.     

Activation of the cGMP signaling pathway is essential in delaying oocyte aging in diabetes mellitus

Uncontrolled diabetes mellitus (DM) adversely affects oocyte maturation and embryo development via mechanisms that are yet unclear. Nonetheless, DM may cause uncoupling of nitric oxide synthases (NOSs) with reduction in the bioavailability of nitric oxide (NO), which is critical to maintain oocyte viability and prevent aging. The current study investigates the role of NO-mediated signaling related to oocyte aging in diabetic and nondiabetic mice. Age-related alterations in the oocytes, including ooplasmic microtubule dynamics (OMD), cortical granule (CG) status, and zona pellucida (ZP) hardening as well as the integrity of the spindle/chromatin were studied using confocal microscopy. Oocytes obtained from diabetic mice exhibited accelerated aging compared to that from nondiabetic mice. Moreover, oocytes from diabetic animals were exquisitely sensitive to NOS and guanylate cyclase (GC) inhibitors (L-NAME, ODQ), which induced aging and relatively resistant to its delay by the cGMP derivative (8-Br-cGMP). Oocytes from nondiabetic control mice displayed similar sensitivity to L-NAME in older oocytes, although to a significantly lower extent than that of DM (P < 0.04-0.0001). Despite the differences in response between DM and nonDM mice, the activation of cGMP pathway is essential to maintain the integrity of oocytes and delay oocyte aging. These findings not only indicate the role of NO signaling in the prevention of oocyte aging but also suggest enhanced aging and NO insufficiency in oocytes from diabetic mice. A comprehensive model incorporating our current findings with NOS, GC, and G kinase cycles is presented.     

Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn²⁺• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd²⁺) and inhibitory (Zn²⁺) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn²⁺.     

Inhibition of renin activity slows down the progression of HIV-associated nephropathy

In the present study, we evaluated the effect of inhibition of renin activity (aliskiren) on the progression of renal lesions in two different mouse models (Vpr and Tg26) of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN). In protocol A, Vpr mice were fed either water (C-VprA) or doxycycline [Doxy (D-VprA)] in their drinking water for 6 wk. In protocols B and C, Vpr mice received either normal saline (C-VprB/C), Doxy + normal saline (D-VprB/C), or Doxy + aliskiren (AD-VprB/C) for 6 wk (protocol B) or 12 wk (protocol C). In protocols D and E, Vpr mice were fed Doxy for 6 wk followed by kidney biopsy. Subsequently, half of the mice were administered either normal saline (D-VprD/E) or aliskiren (AD-VprD/E) for 4 wk (protocol D) or 8 (protocol E) wk. All D-VprA mice showed renal lesions in the form of focal segmental glomerular sclerosis and dilatation of tubules. In protocols B and C, aliskiren diminished both progression of renal lesions and proteinuria. In protocol C, aliskiren also diminished (P < 0.01) the rise in blood urea. In all groups, Doxy-treated mice displayed increased serum ANG I levels (the product of plasma renin activity); on the other hand, all aliskiren-treated mice displayed diminished serum ANG I levels. Renal tissues of D-VprC displayed increased ANG II content; however, aliskiren attenuated renal tissue ANG II production in AD-VprC. In protocol D, AD-VprD showed a 24.2% increase in the number of sclerosed glomeruli compared with 139.2% increase in sclerosed glomeruli in D-VprD (P < 0.01) from their baseline. The attenuating effect of aliskiren on the progression of renal lesions continued in AD-VprE. Aliskiren also diminished blood pressure, proteinuria, and progression of renal lesions in Tg26 mice. These findings indicate that inhibition of renin activity has a potential to slow down the progression of HIVAN.     

Vitamin D receptor activation and downregulation of renin-angiotensin system attenuate morphine-induced T cell apoptosis

Opiates have been reported to induce T cell loss. We evaluated the role of vitamin D receptor (VDR) and the activation of the renin-angiotensin system (RAS) in morphine-induced T cell loss. Morphine-treated human T cells displayed downregulation of VDR and the activation of the RAS. On the other hand, a VDR agonist (EB1089) enhanced T cell VDR expression both under basal and morphine-stimulated states. Since T cells with silenced VDR displayed the activation of the RAS, whereas activation of the VDR was associated with downregulation of the RAS, it appears that morphine-induced T cell RAS activation was dependent on the VDR status. Morphine enhanced reactive oxygen species (ROS) generation in a dose-dependent manner. Naltrexone (an opiate receptor antagonist) inhibited morphine-induced ROS generation and thus, suggested the role of opiate receptors in T cell ROS generation. The activation of VDR as well as blockade of ANG II (by losartan, an AT(1) receptor blocker) also inhibited morphine-induced T cell ROS generation. Morphine not only induced double-strand breaks (DSBs) in T cells but also attenuated DNA repair response, whereas activation of VDR not only inhibited morphine-induced DSBs but also enhanced DNA repair. Morphine promoted T cell apoptosis; however, this effect of morphine was inhibited by blockade of opiate receptors, activation of the VDR, and blockade of the RAS. These findings indicate that morphine-induced T cell apoptosis is mediated through ROS generation in response to morphine-induced downregulation of VDR and associated activation of the RAS.     

Discovery of a novel series of 4-quinolone JNK inhibitors

A novel series of highly selective JNK inhibitors based on the 4-quinolone scaffold was designed and synthesized. Structure based drug design was utilized to guide the compound design as well as improvements in the physicochemical properties of the series. Compound (13c) has an IC₅₀ of 62/170 nM for JNK1/2, excellent kinase selectivity and impressive efficacy in a rodent asthma model.     

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: The case of chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD’s pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin—chelerythrine—acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.