BAFF controls B cell metabolic fitness through a PKCβ- and Akt-dependent mechanism

B cell life depends critically on the cytokine B cell–activating factor of the tumor necrosis factor family (BAFF). Lack of BAFF signaling leads to B cell death and immunodeficiency. Excessive BAFF signaling promotes lupus-like autoimmunity. Despite the great importance of BAFF to B cell biology, its signaling mechanism is not well characterized. We show that BAFF initiates signaling and transcriptional programs, which support B cell survival, metabolic fitness, and readiness for antigen-induced proliferation. We further identify a BAFF-specific protein kinase C β–Akt signaling axis, which provides a connection between BAFF and generic growth factor–induced cellular responses

Cytosol-derived proteins are sufficient for Arp2/3 recruitment and ARF/coatomer-dependent actin polymerization on Golgi membranes

AbstractThe actin cytoskeleton has been implicated in protein trafficking at the Golgi apparatus and in Golgi orientation and morphology. Actin dynamics at the Golgi are regulated in part by recruiting Cdc42 or Rac to the membrane through a binding interaction with the coatomer-coated (COPI)-vesicle coat protein, coatomer. This leads to actin polymerization through the effector, N-WASP and the Arp2/3 complex. Here, we have used reconstitution of vesicle budding to test whether Arp2/3 is recruited to membranes during the formation of COPI vesicles. Our results revealed that ARF1 activation leads to greatly increased Arp3 levels on the membranes. Coatomer-bound Cdc42 and pre-existing F-actin are important for Arp2/3 binding. ARF1-dependent Arp2/3 recruitment and actin polymerization can be reconstituted on liposomal membranes, indicating that no membrane proteins are necessary. These results show that activated ARF1 can stimulate Arp2/3 recruitment to Golgi membranes through coatomer, Cdc42 or Rac, and N-WASP

Nab2p and the Thp1p-Sac3p Complex Functionally Interact at the Interface between Transcription and mRNA Metabolism

THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations. In a search for multicopy suppressors of the transcription defect of thp1Δ cells, we identified the poly(A)+ RNA-binding heterogeneous nuclear ribonucleoprotein Nab2p. Multicopy NAB2 also suppressed the RNA export defect of thp1Δ cells. This result suggests a functional relationship between Thp1p and Nab2p. Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1Δ, although to a minor degree. Reciprocally, a purified His6-tagged Thp1p fusion bound RNA in vitro. In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3Δ confers a transcription defect and hyper-recombination phenotype identical to those of thp1Δ. mRNA degradation was not affected in thp1Δ mutants, implying that their expression defects are not due to mRNA decay. This indicates that Thp1p-Sac3p is a structural and functional unit. Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800–804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621–631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)–coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer–Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation

Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells

Abstract The advent of the multiwavelength anomalousdiffraction phasing methodhas significantlyacceleratedcrystal structuredeterminationand hasbecomethe norm in protein crystallography. This method allowsresearchersto takeadvantageofthe anomalous signal fromdivers eatoms,but thedominantmethod forderivativ epreparation is selenomethionine substitution.S everal generallya pplicable, high-efficiency labeling protocolsh aveb een developed for use in the bacterial, yeast, andbaculovirus/insect cell expressionsystems butnot formammalian tissuec ulture.A sal arge number of proteinso fb iomedical importance cano nlyb ep roducedi ny ields sufficient forX -ray diffraction experimentsi nm ammalian expression systems, it becomesa ll them ore importantt od evelops uchp rotocols.W et herefore evaluateds everal variablest hatp lay roles in determining incorporation levels andr eporth ere as imple protocolf or selenom ethionine modification of proteinsi n mammalian cellsr outinely yielding >90%l abeling efficiency. Keywords: proteinl abeling; proteinc rystallography; selenomethionine; multiplew avelength anomalous diffraction; mammalian cell culture The multiwavelength anomalous diffraction (MAD)phasingm ethod (Hendrickson1 991) hasb ecome them ethod of choice for X-ray phase determination, with >50% of thee xperimentally phased structures deposited in the PDB during thep asty ear beingd etermined by MAD. WhileM AD has allowed researchers to take advantage of thea nomalouss ignal from several diverse heavy atoms, thed ominantm ethodf or heavya tomd erivative preparation is selenomethioninesubstitution. Several factors contribute to the widespread use of selenomethionine substitution, including simplicity, adaptabilitytodifferent expression systems, scalability,a nd, in some cases, an almost quantitative replacement of methionine resulting in ahomogeneousprotein population. This method results in modified proteins withoutsignificantstructural perturbations due to heavy atom incorporation,w hile eliminatingthe difficult and time-consuming screenings forheavy atom derivatives. It is estimated that, foras uccessful MADe xperiment, ones elenomethionine residue is required for every ; 75-100a mino acids (Hendrickson andO gata 1997). This corresponds to ; 80%o fa ll proteins, which have am ethioninecontentof1%ormore (Strub et al.2003). There are twol imitations to the method: First, the calculations abovea ssumeq uantitative( or near-quantitative) methionine substitution, which often is not the case.F or example, as the complexity of the expression system host increases, so does the complexity of the media requiredfor their growth, Article published onlinea head of print. Articlea nd publication date are at http://www.proteinscience.org/cg

Purification and Functional Characterization of a Histone H3-Lysine 4-Specific Methyltransferase

AbstractMethylation of histone H3 at lysine 9 by SUV39H1 and subsequent recruitment of the heterochromatin protein HP1 has recently been linked to gene silencing. In addition to lysine 9, histone H3 methylation also occurs at lysines 4, 27, and 36. Here, we report the purification, molecular identification, and functional characterization of an H3-lysine 4-specific methyltransferase (H3-K4-HMTase), SET7. We demonstrate that SET7 methylates H3-K4 in vitro and in vivo. In addition, we found that methylation of H3-K4 and H3-K9 inhibit each other. Furthermore, H3-K4 and H3-K9 methylation by SET7 and SUV39H1, respectively, have differential effects on subsequent histone acetylation by p300. Thus, our study provides a molecular explanation to the differential effects of H3-K4 and H3-K9 methylation on transcription

The human PAF complex coordinates transcription with events downstream of RNA synthesis.

The yeast PAF (yPAF) complex interacts with RNA polymerase II and coordinates the setting of histone marks associated with active transcription. We report the isolation and functional characterization of the human PAF (hPAF) complex. hPAF shares four subunits with yPAF (hCtr9, hPaf1, hLeo1, and hCdc73), but contains a novel higher eukaryotic-specific subunit, hSki8. RNAi against hSki8 or hCtr9 reduces the cellular levels of other hPAF subunits and of mono- and trimethylated H3-Lys 4 and dimethylated H3-Lys 79. The hSki8 subunit is also a component of the human SKI (hSKI) complex. Yeast SKI complex is cytoplasmic and together with Exosome mediates 3\u27-5\u27 mRNA degradation. However, hSKI complex localizes to both nucleus and cytoplasm. Immunoprecipitation experiments revealed that hPAF and hSKI complexes interact, and ChIP experiments demonstrated that hSKI associates with transcriptionally active genes dependent on the presence of hPAF. Thus, in addition to coordinating events during transcription (initiation, promoter clearance, and elongation), hPAF also coordinates events in RNA quality control

JHDM2A, a JmjC-Containing H3K9 Demethylase, Facilitates Transcription Activation by Androgen Receptor

Covalent modification of histones plays an important role in regulating chromatin dynamics and transcription. Histone methylation was thought to be an irreversible modification until recently. Using a biochemical assay coupled with chromatography, we have purified a JmjC domain-containing protein, JHDM2A, which specifically demethylates mono- and dimethyl-H3K9. Similar to JHDM1, JHDM2A-mediated histone demethylation requires cofactors Fe(II) and alpha-ketoglutarate. Mutational studies indicate that a JmjC domain and a zinc finger present in JHDM2A are required for its enzymatic activity. Overexpression of JHDM2A greatly reduced the H3K9 methylation level in vivo. Knockdown of JHDM2A results in an increase in the dimethyl-K9 levels at the promoter region of a subset of genes concomitant with decrease in their expression. Finally, JHDM2A exhibits hormone-dependent recruitment to androgen-receptor target genes, resulting in H3K9 demethylation and transcriptional activation. Thus, our work identifies a histone demethylase and links its function to hormone-dependent transcriptional activation

Methylation of H3-Lysine 79 Is Mediated by a New Family of HMTases without a SET Domain

AbstractThe N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation [1]. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity [2–4]. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family [3]. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase