Life time—circadian clocks,mitochondria and metabolism

A functional circadian clock has long been considered a selective advantage. Accumulating evidence shows that the clock coordinates a variety of physiological processes in order to schedule them to the optimal time of day and thus to synchronize metabolism to changes in external conditions. In mitochondria, both metabolic and cellular defense mechanisms are carefully regulated. Abnormal clock function, might influence mitochondrial function, resulting in decreased fitness of an organism.     

Glia Maturation Factor (GMF) Interacts with Arp2/3 Complex in a Nucleotide State-dependent Manner

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μm affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.     

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components: BINDING FIDELITY,PROMISCUITY, AND STRUCTURAL BUTTRESSES*

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (K_d = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.     

Structure of the Type VI Effector-Immunity Complex (Tae4-Tai4) Provides Novel Insights into the Inhibition Mechanism of the Effector by Its Immunity Protein

The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The Gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.     

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.