Latest Articles Include:
- The Molecular Dating Game: An Antibody Heavy Chain Hangs Loose with a Chaperone while Waiting for Its Life Partner
- Mol Cell 34(6):635-636 (2009)
In a recent issue of Molecular Cell, Feige et al. (2009) utilize the murine immunoglobulin system to shed light on a long-standing puzzle: how do cells coordinate folding of different polypeptides that ultimately form a complex?
- The Guardian Recruits Cops: The p53-p21 Axis Delegates Prosurvival Duties to the Keap1-Nrf2 Stress Pathway
- Mol Cell 34(6):637-639 (2009)
In this issue of Molecular Cell, Zhang and colleagues (Chen et al., 2009) report their identification of p21 as a positive regulator of Nrf2, which extends p53 prosurvival functions through recruitment of a master stress-response regulator.
- A Histone Code for Regulating V(D)J Recombination
- Mol Cell 34(6):639-640 (2009)
In a recent issue of Molecular Cell, Shimazaki et al. (2009) show that an interaction between RAG2 and a methylated histone might play a critical regulatory role in V(D)J recombination by enhancing DNA binding and enzymatic activity of the V(D)J recombinase.
- The Juxtamembrane Region of the EGF Receptor Functions as an Activation Domain
- Mol Cell 34(6):641-651 (2009)
In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating—rather than autoinhibitory—role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xen! ograft assay.
- KSR2 Is a Calcineurin Substrate that Promotes ERK Cascade Activation in Response to Calcium Signals
- Mol Cell 34(6):652-662 (2009)
Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic β-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new ! mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.
- Direct Interaction between Nrf2 and p21Cip1/WAF1 Upregulates the Nrf2-Mediated Antioxidant Response
- Mol Cell 34(6):663-673 (2009)
In response to oxidative stress, Nrf2 and p21Cip1/WAF1 are both upregulated to protect cells from oxidative damage. Nrf2 is constantly ubiquitinated by a Keap1 dimer that interacts with a weak-binding 29DLG motif and a strong-binding 79ETGE motif in Nrf2, resulting in degradation of Nrf2. Modification of the redox-sensitive cysteine residues on Keap1 disrupts the Keap1-29DLG binding, leading to diminished Nrf2 ubiquitination and activation of the antioxidant response. However, the underlying mechanism by which p21 protects cells from oxidative damage remains unclear. Here we present molecular and genetic evidence suggesting that the antioxidant function of p21 is mediated through activation of Nrf2 by stabilizing the Nrf2 protein. The 154KRR motif in p21 directly interacts with the 29DLG and 79ETGE motifs in Nrf2 and thus competes with Keap1 for Nrf2 binding, compromising ubiquitination of Nrf2. Furthermore, the physiological significance of our findings was demonstrat! ed in vivo using p21-deficient mice.
- Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78
- Mol Cell 34(6):674-685 (2009)
The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, con! sequently, ubiquitylation.
- Glutamine-Specific N-Terminal Amidase, a Component of the N-End Rule Pathway
- Mol Cell 34(6):686-695 (2009)
Deamidation of N-terminal Gln by NtQ-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of NtQ-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants, and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode NtQ-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 NtQ-amidase. Remarkably, "high-throughput" studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases.
- Cellular MicroRNA and P Bodies Modulate Host-HIV-1 Interactions
- Mol Cell 34(6):696-709 (2009)
MicroRNAs (miRNAs), 22 nt noncoding RNAs, assemble into RNA-induced silencing complexes (RISCs) and localize to cytoplasmic substructures called P bodies. Dictated by base-pair complementarity between miRNA and a target mRNA, miRNAs specifically repress posttranscriptional expression of several mRNAs. Here we report that HIV-1 mRNA interacts with RISC proteins and that disrupting P body structures enhances viral production and infectivity. In HIV-1-infected human T lymphocytes, we identified a highly abundant miRNA, miR-29a, which specifically targets the HIV-1 3′UTR region. Inhibiting miR-29a enhanced HIV-1 viral production and infectivity, whereas expressing a miR-29 mimic suppressed viral replication. We also found that specific miR-29a-HIV-1 mRNA interactions enhance viral mRNA association with RISC and P body proteins. Thus we provide an example of a single host miRNA regulating HIV-1 production and infectivity. These studies highlight the significance of miRNAs! and P bodies in modulating host cell interactions with HIV-1 and possibly other viruses.
- Structural Basis of Transcription: Mismatch-Specific Fidelity Mechanisms and Paused RNA Polymerase II with Frayed RNA
- Mol Cell 34(6):710-721 (2009)
We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3′ end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3′ nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.
- Highly Transcribed RNA Polymerase II Genes Are Impediments to Replication Fork Progression in Saccharomyces cerevisiae
- Mol Cell 34(6):722-734 (2009)
Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome-wide approach to identify 96 sites of very high DNA polymerase binding in wild-type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared with wild-type cells, as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are a class of natural pause sites that are not exacerbated in rrm3 cells.
- Crystal Structure of the Rad9-Rad1-Hus1 DNA Damage Checkpoint Complex—Implications for Clamp Loading and Regulation
- Mol Cell 34(6):735-745 (2009)
Rad9, Rad1, and Hus1 form a heterotrimeric complex (9-1-1) that is loaded onto DNA at sites of DNA damage. DNA-loaded 9-1-1 activates signaling through the Chk1 arm of the DNA damage checkpoint response via recruitment and stimulation of ATR. Additionally, 9-1-1 may play a direct role in facilitating DNA damage repair via interaction with a number of DNA repair enzymes. We have now determined the crystal structure of the human 9-1-1 complex, revealing a toroidal structure with a similar architecture to the homotrimeric PCNA DNA-binding clamp. The structure explains the formation of a unique heterotrimeric arrangement and reveals significant differences among the three subunits in the sites implicated in binding to the clamp loader and to ligand proteins. Biochemical analysis reveals a single repair enzyme-binding site on 9-1-1 that can be blocked competitively by the PCNA-binding cell-cycle regulator p21cip1/waf1.
- Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination
- Mol Cell 34(6):746-759 (2009)
Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling! and solution conditions.
- The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli
- Mol Cell 34(6):760-766 (2009)
Ribosome biogenesis is coupled to the transcription of ribosomal (r)RNAs, which enables an ordered and hierarchical assembly of the ribosome starting with the 5′-terminal domain. We constructed four circular permutants (CPs) of Escherichia coli rRNAs in which the original termini of 16S or 23S rRNAs were genetically connected, and new termini were created elsewhere within the same rRNAs (in helix 33 of 16S rRNA, or in helices 45, 63, and 78 of 23S rRNA). Unexpectedly, all CPs tested were able to rescue E. coli strain Δ7 prrn, which lacks all chromosomal rRNA operons. This result demonstrates that hierarchical assembly from the 5′-terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. However, severe growth defects of all CPs were found in the absence of the DEAD box RNA helicase deaD, indicating that DeaD assists in the efficient assembly of each subunit in the cell.
- Shifts in Replication Timing Actively Affect Histone Acetylation during Nucleosome Reassembly
- Mol Cell 34(6):767-774 (2009)
The entire genome is replicated in a programmed manner, with specific regions undergoing DNA synthesis at different times in S phase. Active genes generally replicate in early S phase, while repressed genes replicate late, and for some loci this process is developmentally regulated. Using a nuclear microinjection system, we demonstrate that DNA sequences originally packaged into nucleosomes containing deacetylated histones during late S become reassembled with acetylated histones after undergoing replication in early S. Conversely, a change from early to late replication timing is accompanied by repackaging into nucleosomes containing deacetylated histones. This is carried out by differential cell-cycle-controlled acetylation and deacetylation of histones H3 and H4. These studies provide strong evidence that switches in replication timing may play a role in the regulation of nucleosome structure during development.