Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin.

Calmodulin (CaM) is an important Ca2+ signaling hub that regulates many protein signaling pathways. In recent years, several CaM homologs expressed in plants have been shown to regulate mammalian targets and they are attractive for gene therapy. However, the molecular basis of how the CaM homologs mutations impact target activation is unclear, which limits efforts to engineer their functional properties. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isofroms, sCaM-1 and sCaM-4 share >90% and ≈78% identity with the mammalian CaM (mCaM), respectively, activate CaN with comparable or reduced activity relative to mCaM. We used molecular simulations and experimental assays to probe whether calcium and protein-protein binding interactions are altered in plant CaMs relative to mCaM as a basis for differential CaN regulations. We found that the two sCaMs' calcium binding properties such as coordination and affinity are comparable to mCaM. Further, the binding of CaM to the CaM binding region (CaMBR) in CaN is also comparable among the three CaMs, as evidenced by calculated binding free energies and experimental measured EC50 [CaM]. However, mCaM and sCaM-1 exhibited stronger binding with a secondary region of CaN's regulatory domain that is weakened for sCaM-4. This secondary interaction is likely to affect the turnover rate (kcat) of CaN based on our modeling of enzyme activity and is consistent with our experimental data. Together, our data show how plant-derived CaM variants can alter target activation through interactions beyond calcium binding and canonical CaMBR binding, which may extend beyond the mammalian CaN target.

Synthesis and Characterization of Urethane Side Chain Substituted Diketopyrrolopyrrole

The synthesis of Diketopyrrolopyrrole (DPP) having secondary interaction in the side chain explores its possibility to use in electronic and sensing applications. Herein we report easy method to engineer side chains of DPP. The hydrogen bonding is introduced on the side chain by substitution of urethane side chains on Diketopyrrolopyrrole (DPPurethane).The urethane side chain comprises a branched alkyl chain with good yields and purities. The DPPurethane characterized by NMR and IR, optical properties along with energy minimized structure were studied.

Molecular and Structural Basis of Olfactory Sensory Neuron Coalescence by Kirrel Receptors

ABSTRACTProjections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal IG domains is necessary and sufficient to create homodimers, and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide strong evidence for Kirrel3 homodimerization controlling axonal coalescence.

An enhancer sequence in the intrinsically disordered region of the essential cell division protein FtsZ promotes conformation-guided substrate processing by ClpXP in Escherichia coli

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation by conformationally-guided interactions.

Interactive effects of asparagine and aspartate homeostasis with sex and age for the risk of type 2 diabetes risk

Background Asparagine and aspartate homeostasis are linked with type 2 diabetes (T2D). This study aimed to explore whether asparagine and aspartate metabolism interacted with sex and age to increase the risk of T2D. Methods From 27 May 2015 to 3 August 2016, we consecutively retrieved 1032 T2D patients and 1522 subjects without T2D from a tertiary care hospital in Liaoning, China. Restricted cubic spline nested in the logistic regression was used to draw odds ratio curves of plasma asparagine to aspartate ratio for T2D by sex and age. Cut-off point was selected where curves went apart, indicating possible interaction. Addictive interactions of asparagine to aspartate ratio with sex or age and secondary interaction with copresence of unfavorable sex and age were further estimated using relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (S). Results Ratio of asparagine to aspartate > 1.5 was associated with elevated risk of T2D (OR 7.99, 95%CI 5.50 to 11.6), which was enhanced by female gender to 13.6, (95%CI 8.10–22.9) and by > 50 years of age to 28.7 (14.6–56.3), with significant additive interactions. There was a significant secondary-interaction of copresence of female sex and > 50 years of age with high asparagine to aspartate ratio for increased T2D risk with the OR being further increased to 34.4 (20.5–57.5). Conclusions High asparagine to aspartate ratio was associated with markedly increased risk of T2D, which was further amplified by either female gender or > 50 years of age, and especially both.

Interactive effects of asparagine and aspartate homeostasis with sex and age for the risk of type 2 diabetes risk

Background Asparagine and aspartate homeostasis link with type 2 diabetes (T2D). This study aimed to explore whether asparagine and aspartate metabolism interacted with sex and age to increase the risk of T2D.Methods From 27 May 2015 to 3 August 2016, we consecutively retrieved 1032 T2D patients and 1522 subjects without T2D from a tertiary care hospital in Liaoning, China. Restricted cubic spline nested in the logistic regression was used to draw odds ratio curves of plasma asparagine to aspartate ratio for T2D by sex and age. Cutoff point was select where curves went apart, indicating possible interaction. Addictive interactions of asparagine to aspartate ratio with sex or age and secondary interaction with copresence of unfavorable sex and age were further estimated using relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (S). Results Ratio of asparagine to aspartate >1.5 was associated with elevated risk of T2D (OR: 7.99, 95%CI: 5.50 to 11.6), which was enhanced by female gender to 13.6, (95%CI: 8.10-22.9) and by >50 years of age to 28.7 (14.6-56.3), with significant additive interactions. There was a significant secondary-interaction of copresence of female sex and > 50 years of age with high asparagine to aspartate ratio for increased T2D risk with the OR being further increased to 34.4 (20.5 - 57.5). Conclusions High asparagine to aspartate ratio was associated with markedly increased risk of T2D, which was further amplified by either female gender or >50 years of age, and especially both.

Interactive effects of asparagine and aspartate homeostasis with sex and age for the risk of type 2 diabetes risk

Background Asparagine and aspartate homeostasis link with type 2 diabetes (T2D). This study aimed to explore whether asparagine and aspartate metabolism interacted with sex and age to increase the risk of T2D.Methods From 27 May 2015 to 3 August 2016, we consecutively retrieved 1032 T2D patients and 1522 subjects without T2D from a tertiary care hospital in Liaoning, China. Restricted cubic spline nested in the logistic regression was used to draw odds ratio curves of plasma asparagine to aspartate ratio for T2D by sex and age. Cutoff point was select where curves went apart, indicating possible interaction. Addictive interactions of asparagine to aspartate ratio with sex or age and secondary interaction with copresence of unfavorable sex and age were further estimated using relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (S). Results Ratio of asparagine to aspartate >1.5 was associated with elevated risk of T2D (OR: 7.99, 95%CI: 5.50 to 11.6), which was enhanced by female gender to 13.6, (95%CI: 8.10-22.9) and by >50 years of age to 28.7 (14.6-56.3), with significant additive interactions. There was a significant secondary-interaction of copresence of female sex and > 50 years of age with high asparagine to aspartate ratio for increased T2D risk with the OR being further increased to 34.4 (20.5 - 57.5). Conclusions High asparagine to aspartate ratio was associated with markedly increased risk of T2D, which was further amplified by either female gender or >50 years of age, and especially both.