The tandem BRCT domain of 53BP1 is not required for its repair function

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.     

RAP80 interacts with the SUMO-conjugating enzyme UBC9 and is a novel target for sumoylation

RAP80, a nuclear protein with two functional ubiquitin-interaction motifs (UIMs) at its N-terminus, plays a critical role in the regulation of estrogen receptor alpha and DNA damage response signaling. A yeast two-hybrid screen identified the SUMO-conjugating enzyme UBC9 as a protein interacting with RAP80. The interaction of RAP80 with UBC9 was confirmed by co-immunoprecipitation and GST pull-down analyses. The region between aa 122-204 was critical for the interaction of RAP80 with UBC9. In addition, we demonstrate that RAP80 is a target for SUMO-1 modification in intact cells. Expression of UBC9 enhanced RAP80 mono-sumoylation and also induced multi-sumoylation of RAP80. In addition to SUMO-1, RAP80 was efficiently conjugated to SUMO-3 but was only a weak substrate for SUMO-2 conjugation. These findings suggest that sumoylation plays a role in the regulation of RAP80 functions.     

ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner

Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.     

Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the gamma-H2AX histone tail

MDC1 (mediator of DNA damage checkpoint protein 1) regulates the recognition and repair of DNA double strand breaks in mammalian cells through its interactions with nuclear foci containing the COOH-terminally phosphorylated form of the histone variant, H2AX. Here we demonstrate that the tandem BRCT repeats of MDC1 directly bind to the phosphorylated tail of H2AX-Ser(P)-Gln-Glu-Tyr, in a manner that is critically dependent on the free carboxylate group of the COOH-terminal Tyr residue. We have determined the x-ray crystal structure of the MDC1 BRCT repeats at 1.45 Angstroms resolution. By a comparison with the structure of the BRCA1 BRCT bound to a phosphopeptide, we suggest that two arginine residues in MDC1, Arg(1932) and Arg(1933) may recognize the COOH terminus of the peptide as well as the penultimate Glu of H2AX, while Gln(2013) may provide additional specificity for the COOH-terminal Tyr.     

The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner

Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5′ terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5′-GUAAC / 3′-CUUAG double-stranded motif, which closely resembles the canonical 5′-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar K_D values) that leads to oligomerization of the RBD on its RNA ligands.     

Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains

Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin "marks" on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein.     

Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.     

The polycystin-1 C-type lectin domain binds carbohydrate in a calcium-dependent manner, and interacts with extracellular matrix proteins in vitro

Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expressed the PKD1 C-type lectin domain, and have demonstrated that it binds carbohydrate matrices in vitro, and that Ca(2+) is required for this interaction. This domain also binds to collagens type I, II and IV in vitro. This binding is greatly enhanced in the presence of Ca(2+) and can be inhibited by soluble carbohydrates such as 2-deoxyglucose and dextran. These results suggest that polycystin-1 may be involved in protein-carbohydrate interactions in vivo. The data presented indicate that there may a direct interaction between the PKD1 gene product and an ubiquitous extracellular matrix (ECM) protein.     

Ancient conserved domain protein-1 binds copper and modifies its retention in cells

The ancient conserved domain protein (ACDP) family are a recently identified group of homologous mammalian proteins. Some family members have been suggested to have roles in the metabolism of metals. We investigated the capacity of ACDP-1 to bind metals. Using immobilised metal affinity chromatography and isothermal titration calorimetry we determined that ACDP-1 is a high affinity copper binding protein able to bind copper at nanomolar concentrations. In addition the promoter of ACDP-1 contains metal response elements and the cellular expression of ACDP-1 alters cellular retention of copper. However, cellular expression of ACDP-1 does not alter cellular resistance to the toxicity of copper or other metals. As our findings place the subcellular localisation of ACDP-1 in the cytoplasm it is possible that ACDP-1 represent a novel copper chaperone or storage protein.     

Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.     

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-kappaB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-kappaB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.     

Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response

MOTIVATION: Human Nbs1 and its homolog Xrs2 in Saccharomyces cerevisiae are part of the conserved MRN complex (MRX in yeast) which plays a crucial role in maintaining genomic stability. NBS1 corresponds to the gene mutated in the Nijmegen breakage syndrome (NBS) known as a radiation hyper-sensitive disease. Despite the conservation and the importance of the MRN complex, the high sequence divergence between Nbs1 and Xrs2 precluded the identification of common domains downstream of the N-terminal Fork-Head Associated (FHA) domain. RESULTS: Using HMM-HMM profile comparisons and structure modelling, we assessed the existence of a tandem BRCT in both Nbs1 and Xrs2 after the FHA. The structure-based conservation analysis of the tandem BRCT in Nbs1 supports its function as a phosphoserine binding domain. Remarkably, the 5 bp deletion observed in 95% of NBS patients cleaves the tandem at the linker region while preserving the structural integrity of each BRCT domain in the resulting truncated gene products.     

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.     

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1^−/− mice lack any obvious limb or skeletal defects, Sost^−/− mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost^−/−; Sostdc1^−/− mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost^−/− and Sost^−/−; Sostdc1^−/− mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.     

RAP80 protein is important for genomic stability and is required for stabilizing BRCA1-A complex at DNA damage sites in vivo

RAP80 (receptor-associated protein 80) is a ubiquitin-binding protein that can specifically recognize and bind to Lys-63-linked polyubiquitin chains, thus targeting the BRCA1-A complex to DNA damage sites. To study the role of RAP80 in vivo, we generated RAP80-deficient mice. The deficient mice are prone to B-cell lymphomagenesis. B-cell lymphomas in RAP80-deficient mice are nearly diploid but harbor clonal chromosome translocations. Moreover, the deficient mice are hypersensitive to ionizing radiation. Repair of ionizing radiation-induced DNA double-strand breaks is impaired in RAP80-deficient mouse embryonic fibroblasts. Mechanistically, loss of RAP80 suppresses recruitment of the BRCA1-A complex to DNA damage sites and abrogates the DNA damage repair process at DNA damage sites. Taken together, these results reveal that RAP80 plays a crucial role in the DNA damage response and in maintaining genomic integrity.