Inhibition of CASK Expression by Virus-mediated RNA Interference in Medial Prefrontal Cortex Affects Social Behavior in the Adult Mouse

Article信州医学雑誌 69(1): 45-52(2021)journal articl

Using Protein Homology Models for Structure-Based Studies: Approaches to Model Refinement

Homology modeling is a computational methodology to assign a 3-D structure to a target protein when experimental data are not available. The methodology uses another protein with a known structure that shares some sequence identity with the target as a template. The crudest approach is to thread the target protein backbone atoms over the backbone atoms of the template protein, but necessary refinement methods are needed to produce realistic models. In this mini-review anchored within the scope of drug design, we show the validity of using homology models of proteins in the discovery of binders for potential therapeutic targets. We also report several different approaches to homology model refinement, going from very simple to the most elaborate. Results show that refinement approaches are system dependent and that more elaborate methodologies do not always correlate with better performances from built homology models

Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo

Shape-selective recognition of nucleic acid structures by supramolecular drugs offers the potential to treat disease. The Trans Activation Response (TAR) region is a region of high secondary structure within the human immunodeficiency virus-1 (HIV-1) RNA that complexes with the virus-encoded Transactivator protein (TAT) and regulates viral transcription. Herein, we explore different metallo-supramolecular triple stranded helicates (cylinders) that target the TAR bulge motif and inhibit the formation of TAR-TAT complexes and HIV infection. Cylinders that incorporate Ni(II) and Ru(II) showed the most potent anti-viral activity with limited evidence of cellular cytotoxicity. These metallo-supramolecular compounds provide an exciting avenue for developing a new class of anti-viral agents

Cdc42 localized in the CatSper signaling complex regulates cAMP‐dependent pathways in mouse sperm

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+-dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. State University of Colorado - Fort Collins; Estados UnidosFil: Gervasi, María G.. University of Massachussets; Estados UnidosFil: Orta, Gerardo. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: De la Vega Beltrán, José L.. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: Stival, Cintia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gilio, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: D'alotto Moreno, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados UnidosFil: Darszon, Alberto. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

The Cul3 Ubiquitin Ligase: an Essential Regulator of Diverse Cellular Processes

Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3\u27s role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway

Probing the Role of Nascent Helicity in Protein Function: p27kip1 as a Regulator of the Cell Cycle

p27kip1 binds to and regulates the activity of cyclin-dependent kinases (Cdks) which are the master timekeepers of the cell division cycle. Members of the p27 family of proteins, also including p21 and p57, are called cyclin-dependent kinase inhibitors (CKIs). The amino terminal domain of p27 inhibits Cdk activity and is referred to as the kinase inhibitory domain (KID). The KID is comprised of a cyclin-binding domain (D1) and a Cdk binding domain (D2) joined by a 22 residue linker domain (LH). Structural analysis of the KID in solution before binding its Cdk targets revealed that D1 and D2 are largely unstructured and that the linker domain exhibits nascent helical characteristics. The nascent helical structure of the linker domain is conserved amongst the CKI proteins and I hypothesize that it is an important determinant of their functional properties. To test this hypothesis, I probed the interactions of peptides corresponding to D1 and D2 (LH domain deletion mutant) with the Cdk2/cyclin A complex. Results from isothermal titration calorimetry (ITC) and kinase inhibition assays, show that the interaction and of either D1 or D2 with the Cdk2/cyclin A complex is less favorable relative to that of the p27-KID and that the D2 peptide cannot completely inhibit Cdk2/cyclin A kinase activity. These results indicate that the LH domain that couples D1 and D2 is necessary for the function of p27 as a Cdk inhibitor. Despite having a conserved structure, the linker domains of the Cip/Kip proteins do not have a conserved sequence. However, the sequence of this domain in each Cip/Kip protein in different species is well conserved suggesting that sequence may play a role in the function of these proteins. I explored this sequence-divergence/structure-conservation relationship of the linker domains of the Cip/Kip proteins by constructing chimeric p27-KID molecules in which the p27 linker domain was replaced with the corresponding segments of either p21 or p57. ITC and kinase inhibition assay results show that the chimeric molecules bind and inhibit Cdk2/cyclin A in a manner similar to that of p27-KID. Moreover, thermal denaturation studies show that the complexes formed by these proteins bound to Cdk2/cyclin A have comparable melting temperatures. Taken together, these results indicate that the different linker domains, despite their structural differences, play similar structural roles in p27 binding to Cdk2/cyclin A. In a third study, I investigated the consequences of perturbing the conserved structure of the linker domain. I successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. The secondary structural properties of the mutants were characterized by circular dichroism spectropolarimetry (CD). Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/cyclin A were less stable compared to the ternary complex formed by wild- type p27(p27-KIDwt). Thermodynamic analysis showed that there was a decrease in the enthalpy of association of the mutants with Cdk2/cyclin A relative to p27-KIDwt. The free energies of binding varied within a much narrower range. In addition, in vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Further, when we over-expressed the p27 variants in mouse fibroblasts, they were less effective in causing cell cycle arrest relative to wild-type p27. These results indicate that the conserved structure of the linker domain is important for p27 function. It seems that nature has selected the sequence of the p27 LH domain to be partially structured and that bolstering or eliminating this structure is deleterious to function; intrinsic structure is critical for function

Keap1 modulates the redox cycle and hepatocyte cell cycle in regenerating liver

Keap1 negatively controls the activity of transcription factor Nrf2. This Keap1/Nrf2 pathway plays a critical role in combating oxidative stress. We aimed at determining whether and how Keap1 modulates the cell cycle of replicating hepatocytes during liver regeneration. Two-thirds partial hepatectomy (PH) was performed on wild-type mice and Keap1+/- (Keap1 knockdown) mice. We found that, following PH, Keap1 knockdown resulted in a delay in S-phase entry, disruption of S-phase progression, and loss of mitotic rhythm of replicating hepatocytes. These events are associated with dysregulation of c-Met, EGFR, Akt1, p70S6K, Cyclin A2, and Cyclin B1 in regenerating livers. Astonishingly, normal regenerating livers exhibited the redox fluctuation coupled with hepatocyte cell cycle progression, while keeping Nrf2 quiescent. Keap1 knockdown caused severe disruption in both the redox cycle and the cell cycle of replicating hepatocytes. Thus, we demonstrate that Keap1 is a potent regulator of hepatic redox cycle and hepatocyte cell cycle during liver regeneration

Synthesis and binding studies of peptide mimetics, anion receptors, and kinase inhibitors

A series of novel purine-based fluoroaryl triazoles were synthesized and assayed for their neuroprotective effects of hippocampal slice culture exposed to amyloid beta (Aß) oligomers. A monofluorinated triazole has been identified which has comparable neuroprotective effect as that of flavopiridol and roscovitine against the Aß induced neurotoxicity. Carnosine and histidine are biologically interesting antioxidants. In order to probe whether they exert their antioxidant effect through metal ion chelation, the Cu(ll) ion chelating abilities of these compounds were measured by UV-vis spectroscopy. Amyloid beta, the major component of senile plaques in Alzheimer\u27s disease, is known to complex transition metal ions through histidine residues. In this study, using ¹H NMR titration experiments, it was shown that histidine binds strongly to Zn(II), Cu(Il), and Fe(III) ions at a biologically relevant pH (pH 7.4), with a stoichiometry of Zn(II): histidine binding of 1:2. Fluorinated boroxines, tris(2,6-difluorophenyl)boroxin (DF), tris(2,4,6- trifluorophenyl)boroxin (TF), and tris(pentafluorophenyl)boroxin (PF) were synthesized and investigated for their fluoride anion binding affinity using multinuclear Nuclear magnetic resonance (NMR) and tandem mass spectroscopic techniques. DFT calculations show that the fluoride ion complex of DF prefers unsymmetrical, covalently bound structure over the symmetrically bridged species by 12.5 kcal/mol --Abstract, page iii