Volume 18 Issue 7, July 2011

Energy coupling factor (ECF) transporters consist of an integral membrane protein that confers substrate specificity (S-component) and an energizing module related to ATP-cassette (ABC) transporters. Structural studies of the thiamin-specific S-component ThiT from Lactococcus lactis reveal an interaction with the energizing module through a conserved motif on the membrane-embedded surface of ThiT, thereby providing insight into the substrate translocation mechanism.

RNF8 is an E3 ligase that functions in the DNA damage response by promoting the ubiquitination of histone proteins at double-strand breaks. Now, analysis of mice with a double knockout of RNF8 and related E3 ligase Chfr reveals that these two proteins collaborate to activate ATM via ubiquitination of H2B and consequent recruitment of MRG15, a component of HAT complexes, resulting in histone H4 Lys16 acetylation and chromatin relaxation.

ATR-X syndrome is a congenital disease linked to mutations in ATRX. The ADD domain in ATRX is now found to form a new histone H3 recognition module with binding being promoted by H3K9me3 and antagonized by H4K4me3. Co-crystal structures indicated that H3K9me3 is bound in a polar pocket formed between a GATA-like and PHD domain. Loss of this binding leads to ATRX mislocalization in vivo and has been linked to ATR-X syndrome.

Mutations in the ADD domain of ATRX protein lead to severe mental retardation. Now the importance of this domain for targeting ATRX to heterochromatin is examined; ADD simultaneously recognizes unmethylated lysine 4 and trimethylated lysine 9 of histone H3, making it a combinatorial reader. This readout is enhanced by interaction with HP-1, forming a tripartite network that may bridge adjacent nucleosomes.

Myosin X is involved in cytoskeletal processes including the extension of filopodia. It is now found that the tail of this myosin can inhibit motor activity in a manner that is antagonized by binding of PIP3, which also promotes dimer formation. Interfering with PIP3 binding affects myosin X translocation in vivo, suggesting that lipids regulate the activity of this motor.

The protein OCRL is linked to Lowe syndrome and Dent disease, two related diseases. Mutations in the OCRL Ash-RhoGAP domain abolish its interactions with the F&H motifs in APPL1, Ses1 and Ses2. Structural and biochemical analysis of the OCRL Ash-RhoGAP domain with F&H motifs shows that clinical mutations destabilize the Ash-RhoGAP domain, abolishing the interactions between OCRL- and F&H-motif-containing proteins.

G-quadruplexes form from G rich sequences and have previously been suggested to be involved in various aspects of mRNA metabolism. The structure of an RNA quadruplex-duplex junction in complex with an Arg-Gly-rich peptide from Fragile X mental retardation protein (FMRP) now reveals an unprecedented RNA scaffold and principles underlying its specific recognition.

Synaptotagmin-1 is known to accelerate membrane fusion during neuronal exocytosis in response to Ca²⁺, but how it does this is unclear. By probing the activity of synaptotagmin-1 under conditions of low ionic strength, it is now shown that SNARE-mediated fusion is dependent on synaptotagmin-1, which tethers liposomes together but at distances too far for fusion. Ca²⁺ then induces synaptotagmin-1 to bring the liposomes closer together, allowing fusion to proceed.

Synaptotagmin-1 aggregates membranes in response to Ca²⁺. Using a variety of approaches, it was discovered that synaptotagmin-mediated membrane aggregation occurs through trans interactions between synaptotagmin molecules bound to different membranes. Synaptotagmin-regulated, SNARE-catalyzed fusion further requires synaptotagmin to drive the assembly of nonfusogenic syntaxin and SNAP25 into fusion-competent t-SNARE heterodimers.

The TARDBP gene encodes TDP-43, a multifunctional DNA- and RNA-binding protein involved in many cellular processes. Mutations in TARDBP are associated with TDP-43 proteinopathies. In vivo and in vitro studies of mutants and peptides show similarities between TDP-43 and prion proteins, suggesting that TDP-43 derivatives may cause disease by spreading to neighboring neurons.

Heterochromatin factor KAP-1 is phosphorylated by ATM during the DNA damage response. Now the functional consequences of this modification are explored, revealing that the phosphorylated C-terminal region of KAP-1 perturbs the interaction between auto-SUMOylated KAP-1 and CHD3/Mi-2a, a component of the chromatin remodeler NuRD. This results in CHD3 dispersion from heterochromatin and allows DNA repair to proceed.

Myotonic Dystrophy (DM), the most common muscular dystrophy in adults, is an RNA gain-of-function disease caused by expanded CUG or CCUG repeats that sequester the RNA binding protein MBNL1. MBNL1 is now shown to regulate pre-miR-1 processing. In DM patients, MBNL1 levels are low and another protein binds to pre-miR-1 and promotes its subsequent uridylation, making it resistant to Dicer cleavage. This results in lower amounts of miR-1 and increased levels of its targets, GJA1 and CACNA1C, that encode the main calcium and gap junction channels in the heart, respectively. Thus their misregulation may contribute to the cardiac dysfunctions observed in DM patients.

The RGS family is involved in regulating G protein signaling. Using computational analysis coupled with examination of RGS activity, a group of variable residues that together modulate G protein recognition are now identified. Mutational analysis confirmed the importance of these modulatory residues and generated gain-of-function RGS proteins. The analysis described is shown to be applicable to uncovering important residues in other protein families.

Eisosomes are essential for plasma membrane organization in yeast. Structural, biochemical and cell biological studies now reveal that the main eisosome components are part of the conserved BAR-domain family of proteins, which bind and shape membranes.

Ash2L is part of a complex involved in histone H3 lysine 4 trimethylation, linked to active transcription. Ash2L is now found to contain a helix-wing-helix DNA binding domain that is needed for targeting and H3K4 trimethylation at the β-globin locus control region in vivo.