A genetic strategy for probing the functional diversity of magnetosome formation.

Model genetic systems are invaluable, but limit us to understanding only a few organisms in detail, missing the variations in biological processes that are performed by related organisms. One such diverse process is the formation of magnetosome organelles by magnetotactic bacteria. Studies of model magnetotactic α-proteobacteria have demonstrated that magnetosomes are cubo-octahedral magnetite crystals that are synthesized within pre-existing membrane compartments derived from the inner membrane and orchestrated by a specific set of genes encoded within a genomic island. However, this model cannot explain all magnetosome formation, which is phenotypically and genetically diverse. For example, Desulfovibrio magneticus RS-1, a δ-proteobacterium for which we lack genetic tools, produces tooth-shaped magnetite crystals that may or may not be encased by a membrane with a magnetosome gene island that diverges significantly from those of the α-proteobacteria. To probe the functional diversity of magnetosome formation, we used modern sequencing technology to identify hits in RS-1 mutated with UV or chemical mutagens. We isolated and characterized mutant alleles of 10 magnetosome genes in RS-1, 7 of which are not found in the α-proteobacterial models. These findings have implications for our understanding of magnetosome formation in general and demonstrate the feasibility of applying a modern genetic approach to an organism for which classic genetic tools are not available.

Analysis of host gene expression changes reveals distinct roles for the cytoplasmic domain of the Epstein-Barr virus receptor/CD21 in B-cell maturation, activation, and initiation of virus infection.

UNLABELLED: Epstein-Barr virus (EBV) attachment to human CD21 on the B-cell surface initiates infection. Whether CD21 is a simple tether or conveys vital information to the cell interior for production of host factors that promote infection of primary B cells is controversial, as the cytoplasmic fragment of CD21 is short, though highly conserved. The ubiquity of CD21 on normal B cells, the diversity of this population, and the well-known resistance of primary B cells to gene transfer technologies have all impeded resolution of this question. To uncover the role(s) of the CD21 cytoplasmic domain during infection initiation, the full-length receptor (CD21=CR), a mutant lacking the entire cytoplasmic tail (CT), and a control vector (NEO) were stably expressed in two pre-B-cell lines that lack endogenous receptor. Genome-wide transcriptional analysis demonstrated that stable CD21 surface expression alone (either CR or CT) produced multiple independent changes in gene expression, though both dramatically decreased class I melanoma-associated antigen (MAGE) family RNAs and upregulated genes associated with B-cell differentiation (e.g., C2TA, HLA-II, IL21R, MIC2, CD48, and PTPRCAP/CD45-associated protein). Temporal analysis spanning 72 h revealed that not only CR- but also CT-expressing lines initiated latency. In spite of this, the number and spectrum of transcripts altered in CR- compared with CT-bearing lines at 1 h after infection further diverged. Differential modulation of immediate early cellular transcripts (e.g., c-Jun and multiple histones), both novel and previously linked to CD21-initiated signaling, as well as distinct results from pathway analyses support a separate role for the cytoplasmic domain in initiation of intracellular signals. IMPORTANCE: Membrane proteins that mediate virus attachment tether virus particles to the cell surface, initiating infection. In addition, upon virus interaction such proteins may transmit signals to the interior of the cell that support subsequent steps in the infection process. Here we show that expression of the Epstein-Barr virus B-cell attachment receptor, CD21, in B cells that lack this receptor results in significant changes in gene expression, both before and rapidly following EBV-CD21 interaction. These changes translate into major signaling pathway alterations that are predicted to support stable infection.

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.

Kaposi sarcoma is considered a neoplasm of lymphatic endothelium infected with Kaposi sarcoma-associated herpesvirus. It is characterized by the expression of lymphatic lineage-specific genes by Kaposi sarcoma tumor cells. Here we show that infection of differentiated blood vascular endothelial cells with Kaposi sarcoma-associated herpesvirus leads to their lymphatic reprogramming; induction of approximately 70% of the main lymphatic lineage-specific genes, including PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes.

Discovery and Structure-Based Design of Inhibitors of the WD Repeat-Containing Protein 5 (WDR5)-MYC Interaction.

WDR5 is a critical chromatin cofactor of MYC. WDR5 interacts with MYC through the WBM pocket and is hypothesized to anchor MYC to chromatin through its WIN site. Blocking the interaction of WDR5 and MYC impairs the recruitment of MYC to its target genes and disrupts the oncogenic function of MYC in cancer development, thus providing a promising strategy for the treatment of MYC-dysregulated cancers. Here, we describe the discovery of novel WDR5 WBM pocket antagonists containing a 1-phenyl dihydropyridazinone 3-carboxamide core that was identified from high-throughput screening and subsequent structure-based design. The leading compounds showed sub-micromolar inhibition in the biochemical assay. Among them, compound 12 can disrupt WDR5-MYC interaction in cells and reduce MYC target gene expression. Our work provides useful probes to study WDR5-MYC interaction and its function in cancers, which can also be used as the starting point for further optimization toward drug-like small molecules.

Discovery of Potent Small-Molecule Inhibitors of WDR5-MYC Interaction.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes. It is also a prominent target for pharmacological inhibition in diseases such as cancer, aging, and neurodegenerative disorders. Interactions between WDR5 and various partners are essential for sustaining its function. Most drug discovery efforts center on the WIN (WDR5 interaction motif) site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM (WDR5 binding motif) pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization, and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy.

The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9.

The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a ;hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity.

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

Paleomagnetism. Solar nebula magnetic fields recorded in the Semarkona meteorite.

Magnetic fields are proposed to have played a critical role in some of the most enigmatic processes of planetary formation by mediating the rapid accretion of disk material onto the central star and the formation of the first solids. However, there have been no experimental constraints on the intensity of these fields. Here we show that dusty olivine-bearing chondrules from the Semarkona meteorite were magnetized in a nebular field of 54 ± 21 microteslas. This intensity supports chondrule formation by nebular shocks or planetesimal collisions rather than by electric currents, the x-wind, or other mechanisms near the Sun. This implies that background magnetic fields in the terrestrial planet-forming region were likely 5 to 54 microteslas, which is sufficient to account for measured rates of mass and angular momentum transport in protoplanetary disks.

A Smac mimetic rescue screen reveals roles for inhibitor of apoptosis proteins in tumor necrosis factor-alpha signaling.

Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFalpha signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFalpha induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFalpha-mediated apoptosis.

The crystal structure of human CD21: Implications for Epstein-Barr virus and C3d binding.

Human complement receptor type 2 (CD21) is the cellular receptor for Epstein-Barr virus (EBV), a human tumor virus. The N-terminal two short consensus repeats (SCR1-SCR2) of the receptor interact with the EBV glycoprotein gp350/220 and also with the natural CD21 ligand C3d. Here we present the crystal structure of the CD21 SCR1-SCR2 fragment in the absence of ligand and demonstrate that it is able to bind EBV. Based on a functional analysis of wild-type and mutant CD21 and molecular modeling, we identify a likely region for EBV attachment and demonstrate that this region is not involved in the interaction with C3d. A comparison with the previously determined structure of CD21 SCR1-SCR2 in complex with C3d shows that, in both cases, CD21 assumes compact V-shaped conformations. However, our analysis reveals a surprising degree of flexibility at the SCR1-SCR2 interface, suggesting interactions between the two domains are not specific. We present evidence that the V-shaped conformation is induced by deglycosylation of the protein, and that physiologic glycosylation of CD21 would result in a more extended conformation, perhaps with additional epitopes for C3d binding.

Characterization of Kaposi sarcoma-associated herpesvirus/human herpesvirus-8 infection of human vascular endothelial cells: early events.

Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) is causally associated with Kaposi sarcoma (KS). The absence of a cell culture system that effectively reproduces the composite mechanisms governing initiation and maintenance of HHV-8 infection (lytic and latent) in KS endothelial cells, however, has left important questions unanswered. Here, we report a culture system in which the earliest events that accompany HHV-8 infection could be surveyed in primary endothelial cells. Binding of HHV-8 to microvascular dermal endothelial cells (MVDECs) was directly compared with other primary target cells implicated in HHV-8-associated diseases. Virus attachment, fusion, internalization and transport within MVDECs was monitored by electron microscopy. Studies of genome configuration revealed that rapid circularization of the viral DNA occurred on entry, though by 72 hours after infection linear DNAs accumulated and early as well as late lytic RNAs (T1.1, K8.1) could be detected. The latency transcripts (LT1/LT2) were first detected on day 8, demonstrating that both lytic and latent infection were initiated. Although most lytic transcripts accrued until passage, open-reading frame-74 RNAs fluctuated with a fixed periodicity, suggesting that early replication after infection of MVDECs was synchronous.

EBV attachment stimulates FHOS/FHOD1 redistribution and co-aggregation with CD21: formin interactions with the cytoplasmic domain of human CD21.

CD21 is a multifunctional receptor for Epstein-Barr virus (EBV), for C3dg and for CD23. Upon engagement of immune complexes CD21 modulates immunoreceptor signaling, linking innate and adaptive immune responses. The mechanisms enabling CD21 to independently relay information between the exterior and interior of the cell, however, remain unresolved. We show that formin homologue overexpressed in spleen (FHOS/FHOD1) binds the cytoplasmic domain of human CD21 through its C terminus. When expressed in cells, EGFP-FHOS localizes to the cytoplasm and accumulates with actin in membrane protrusions. Plasma membrane aggregation, redistribution and co-localization of both proteins are stimulated when EBV (ligand) binds CD21. Though widely expressed, FHOS RNA is most abundant in the littoral cell, a major constituent of the red pulp of human spleen believed to function in antigen filtration. Formins are molecular scaffolds that nucleate actin by a pathway distinct from Arp2/3 complex, linking signal transduction to actin reorganization and gene transcription. Thus, ligand stimulation of FHOS-CD21 interaction may transmit signals through promotion of cytoskeletal rearrangement. Moreover, formin recruitment to sites of actin assembly initiated by immunoreceptors could be a general mechanism whereby co-receptors such as CD21 modulate intracellular signaling.