Volume 20 Issue 4, April 2013

Macrodomains are conserved globular domains that can interact with, and in some cases modify, ADP-ribose–based molecules. In this issue, two reports add to the functional repertoire of this domain, by demonstrating that a subset of macrodomain-containing proteins functions to catalyze the removal of protein-proximal ADP-ribose.

The three-dimensional structure of a key complex in chaperone-mediated protein disaggregation, comprising ClpB and DnaK, has been determined using NMR. In addition to providing unique mechanistic insights, the approach promises to elucidate the structural basis for the assembly of elusive dynamic protein machineries in the near future.

To ensure replication success, DNA polymerases must negotiate encounters with actively transcribing RNA polymerases that share the genome. This Review highlights the strategies used by prokaryotic and eukaryotic cells to minimize the consequences of collisions between replication forks and transcription complexes to effect faithful DNA replication without compromising gene expression.

G protein–coupled receptors (GPCRs) mediate transmembrane signaling. The crystal structure of β1-adrenergic receptor in its ligand-free, basal state is now presented. The structure reveals an inactive conformation with two alternating dimer interfaces.

While the cytoplasmic functions of actin are well established, its nuclear roles remain poorly understood. Genetic and biochemical analyses of the nuclear actin-containing yeast INO80 chromatin-remodeling complex now demonstrate that actin functions as a monomer in the nucleus and contributes to INO80 remodeling activity, suggesting a mechanism fundamentally different from that of cytoplasmic actin.

In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates Atg8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.

The PTEN tumor-suppressor gene is regulated by the PTENpg1 long noncoding RNA. A new study investigating a previously uncharacterized PTENpg1-encoded antisense RNA (asRNA) now reveals that the asRNA α isoform functions as a negative regulator of PTEN transcription by recruiting chromatin remodelers and catalyzing H3K27me3 formation, whereas the asRNA β isoform interacts with PTENpg1, thereby affecting PTEN levels post-transcriptionally.

SIX1–EYA form a bipartite transcription factor, essential for development, whose overexpression is linked to metastasis. The crystal structure of SIX1 bound to EYA2 reveals that the interaction is mediated by an α-helix of SIX1, and a single mutation can disrupt this association and SIX1's ability to promote EMT and metastasis.

EM analyses reveal the three-dimensional structure of human telomerase. Along with functional biochemistry data, the work reveals that telomerase is dimeric in its active form. Each of the two TERT subunits can bind telomeric DNA substrates, and both active sites are required for function.

Defective mismatch-repair function is associated with hereditary nonpolyposis colorectal cancer (HNPCC), and mutations in the MLH1 subunit of the MutLα heterodimer MLH1–PMS1 underlie half of HNPCC cases. Structural analysis of the conserved C-terminal domain of Mlh1 from budding yeast shows that Mlh1 contributes to the functionally important Pms1 endonuclease site and provides a rationale for the deleterious impact of MLH1 mutations.

The inhibitory protein SOCS3 plays a key part in hematopoiesis by regulating signaling induced by specific cytokines. The crystal structure of SOCS3 bound to JAK2 and a fragment of the interleulkin-6 receptor reveals how SOCS3 targets specific receptor–JAK complexes and how it exerts its inhibitory activity by blocking substrate binding.

NMDA receptors are heterotetrameric ligand-gated ion channels, with each subunit containing two extracellular clamshell-like domains. The dynamics of GluN1 NTD and its role in NMDA-receptor function are now explored by using functional and computational approaches. GluN1NTD is highly mobile and influences the gating and pharmacological profile of the receptor.

Friedreich's ataxia is one of several hereditary neurodegenerative disorders caused expansion of trinucleotide repeats, but the mechanism of their genomic propagation is unknown. A new plasmid-based system to probe human replicative intermediates reveals that GAA/TTC repeats interfere with replication, thus suggesting that repeat expansion occurs by postreplicative mechanisms.

The protein CPEB, required for learning-related synaptic plasticity in the snail Aplysia, has been suggested to convert from a soluble to a prion-like state. Now these conformational forms of Aplysia CPEB are directly observed, with the prion form showing enhanced binding to target mRNAs.

ADP-ribosylation is an important post-translational protein modification, yet enzymes capable of removing the protein-proximal ADP-ribose were unknown. Human macrodomain proteins MacroD1, D2 and C6orf130 are now shown to catalyze mono-ADP-ribose removal. MacroD2 is also shown to reverse the inhibition of GSK3A caused by ARTD10-catalyzed mono-ADP-ribosylation.

ADP-ribosylation catalyzed by PARPs and sirtuins is an important post-translation modification. Macrodomain proteins MacroD1 and D2 are now shown to preferentially bind mono-ADP-ribosylated proteins and to act as proximal ADP-ribosylhydrolases. The crystal structure of the MacroD2–ADPr complex suggests a catalytic mechanism for the reaction.

Human Staufen1 (STAU1) functions in mRNA decay and other cellular processes. Structural and functional studies reveal that a new, conserved motif together with a degenerate double-stranded RNA–binding domain mediate STAU1 dimerization through a 'domain-swapping' interaction and that dimerization is important for efficient STAU1-mediated decay in vivo.

SUMOylation is a dynamic protein post-translational modification that regulates many eukaryotic proteins. Now a methodology using commercially available monoclonal antibodies coupled to MS analysis leads to the enrichment and identification of endogenous targets for SUMO1 and for SUMO2/3 in HeLa cells and mouse liver. This protocol can be adapted for other tissues and organs.