Journal of Biological Chemistry

doi: 10.1016/s0021-9258(20)58422-6pmid: N/A

doi: 10.1016/s0021-9258(20)58421-4pmid: N/A

Allis, C. David

doi: 10.1074/jbc.x115.663229pmid: 25944906

Like most, if not all, graduate students, I faced some hurdles along the way that seemed higher than the rest. In my doctoral program at Indiana University in Bloomington, a required, but to me dreaded, seminar course in genetics stood in the way. It was a small class that was directed by a professor well known for demanding painful excellence from all those who enrolled. My self-confidence was shaky, but I knew that I did not want to strike out.

Kalinowska, Magdalena;Chávez, Andrés E.;Lutzu, Stefano;Castillo, Pablo E.;Bukauskas, Feliksas F.;Francesconi, Anna

doi: 10.1074/jbc.m115.640136pmid: 25944910

<p>Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca²⁺-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.</p>

Su, Yan;Patra, Amritraj;Harp, Joel M.;Egli, Martin;Guengerich, F. Peter

doi: 10.1074/jbc.m115.653691pmid: 25947374

<p>Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers <i>in vivo</i>.</p>

Fan, Gaofeng;Aleem, Saadat;Yang, Ming;Miller, W. Todd;Tonks, Nicholas K.

doi: 10.1074/jbc.m115.651703pmid: 25897081

<p>Despite significant evidence to the contrary, the view that phosphatases are "nonspecific" still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as "erasers" that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of "nonspecific phosphatases." We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with <i>both</i> classes of enzymes displaying exquisite specificity.</p>

doi: 10.1074/jbc.p115.651703pmid: N/A

♦ See referenced article, J. Biol. Chem. 2015, 290, 15934–15947

Shields-Cutler, Robin R.;Crowley, Jan R.;Hung, Chia S.;Stapleton, Ann E.;Aldrich, Courtney C.;Marschall, Jonas;Henderson, Jeffrey P.

doi: 10.1074/jbc.m115.645812pmid: 25861985

<p>During <i>Escherichia coli</i> urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with <i>E. coli</i> iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved <i>E. coli</i> siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.</p>

doi: 10.1074/jbc.p115.645812pmid: N/A

♦ See referenced article, J. Biol. Chem. 2015, 290, 15949–15960

Hänelt, Inga;Jensen, Sonja;Wunnicke, Dorith;Slotboom, Dirk Jan

doi: 10.1074/jbc.m115.656876pmid: 25922069

<p>Glt_Ph from <i>Pyrococcus horikoshii</i> is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (<i>K_d</i> 120 mm), with a particularly low <i>k</i>_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (<i>E_a</i> 106.8 kJ mol⁻¹) and consequently has a large <i>Q</i>₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the <i>K_d</i> for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (<i>k</i>_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low <i>E_a</i> and <i>Q</i>₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.</p>