Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.NWO15.002.Metals in Catalysis, Biomimetics & Inorganic MaterialsBio-organic Synthesi

Mammalian MCM Loading in Late-G1 Coincides with Rb Hyperphosphorylation and the Transition to Post-Transcriptional Control of Progression into S-Phase

BACKGROUND: Control of the onset of DNA synthesis in mammalian cells requires the coordinated assembly and activation of the pre-Replication Complex. In order to understand the regulatory events controlling preRC dynamics, we have investigated how the timing of preRC assembly relates temporally to other biochemical events governing progress into S-phase. METHODOLOGY/PRINCIPAL FINDING: In murine and Chinese hamster (CHO) cells released from quiescence, the loading of the replicative MCM helicase onto chromatin occurs in the final 3-4 hrs of G(1). Cdc45 and PCNA, both of which are required for G(1)-S transit, bind to chromatin at the G(1)-S transition or even earlier in G(1), when MCMs load. An RNA polymerase II inhibitor (DRB) was added to synchronized murine keratinocytes to show that they are no longer dependent on new mRNA synthesis 3-4 hrs prior to S-phase entry, which is also true for CHO and human cells. Further, CHO cells can progress into S-phase on time, and complete S-phase, under conditions where new mRNA synthesis is significantly compromised, and such mRNA suppression causes no adverse effects on preRC dynamics prior to, or during, S-phase progression. Even more intriguing, hyperphosphorylation of Rb coincides with the start of MCM loading and, paradoxically, with the time in late-G(1) when de novo mRNA synthesis is no longer rate limiting for progression into S-phase. CONCLUSIONS/SIGNIFICANCE: MCM, Cdc45, and PCNA loading, and the subsequent transit through G(1)-S, do not depend on concurrent new mRNA synthesis. These results indicate that mammalian cells pass through a distinct transition in late-G(1) at which time Rb becomes hyperphosphorylated and MCM loading commences, but that after this transition the control of MCM, Cdc45, and PCNA loading and the onset of DNA replication are regulated at the post-transcriptional level

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase

Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S phase and represents a novel form of replication licensing control

CHO cells do not require <i>de novo</i> mRNA synthesis during late G₁, or for progression through S-phase.

<p>(<i>A</i>) Diagram illustrating the experimental design for the data obtained in <i>B&C</i>. (<i>B&C</i>) CHO cells were synchronized in G₀ by isoleucine deprivation and then released into the cell cycle by re-addition of complete medium. At the times indicated, control cells were pulsed with BrdU to determine the kinetics of progression through G₁ into S-phase (gray columns on left in <i>C</i>; examples shown in <i>B</i>). Cells treated with 50 µM DRB at the times indicated were allowed to progress to the peak of S-phase at 12 hrs, at which time they were pulsed with BrdU to determine the percentage of cells that could enter S-phase following different times of DRB exposure (black columns on right in <i>C</i>; examples shown in <i>B</i>). As a control, the DMSO carrier was added to a parallel culture at 1 hr and remained until the BrdU pulse at 12 hrs (white column on right in <i>C</i>). The 12 hr untreated control (gray column on right in <i>C</i>) indicates the maximum number of BrdU-labeled cells obtained without drug treatment. The means of triplicate counts of ∼200 cells/field+/−1 s. d. are shown. (<i>D</i>) RT-PCR analysis of c-<i>myc</i> mRNA levels on samples collected at the indicated times, with and without DRB exposure at 8 hrs. (<i>E</i>) Synchronized CHO cells were untreated (control, top row), or treated with 50 µM DRB at 8 hrs (bottom row), and pulsed with BrdU at each time point indicated in order to measure progression into and through S-phase. At least three fields of ∼200 cells were scored, and averages are displayed in panels with representative fields. Standard deviations (not shown) were within 1–5% for all panels. (<i>F</i>) Parallel to the samples in <i>E</i>, cells were collected and processed by flow cytometry using PI staining.</p

MCM, Cdc45, and PCNA load in the final 3 hrs of G₁ in CHO cells.

<p>(<i>A</i>) Parallel to the BrdU and flow cytometry collection in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005462#pone-0005462-g003" target="_blank">Figure 3 E&F</a>, CHO cells (half treated with DRB at 8 hrs) were collected and separated into total cell lysates (TCE), or fractionated into nucleosolic/cytosolic detergent-soluble extracts (S1) or chromatin-bound detergent-resistant extracts (P3). Immunoblotting with the indicated antibodies was performed on lysates from equal cell numbers loaded into each lane. The G₁-S transition in CHO cells (9 hrs after release) is overlayed in gray. (<i>B</i>) An enlargement of the time points from part <i>A</i> for hours G₀ through 9 is shown for Mcm2 and Mcm5 immunoblots.</p

Summary of results described in this report.

<p>Mammalian cells require ongoing mRNA synthesis in the first part of G₁-phase. Concurrent with this timeframe, the Rb protein is hypophosphorylated and MCMs have not loaded onto chromatin at the preRCs. In the final 3–4 hours of G₁-phase, mammalian cells pass through a transition when Rb is hyperphosphorylated, MCMs load onto chromatin, and new mRNA synthesis is no longer rate-limiting for MCM, Cdc45, or PCNA loading, nor for the eventual progression of the cells into and through S-phase.</p

MCM, Cdc45, and PCNA load in the final 4 hrs of G₁ in Balb/MK cells.

<p>(<i>A</i>) BrdU was pulsed into MK cells at the indicated times following release from quiescence to determine the kinetics of synchronization and entry into S-phase. (<i>B</i>) In parallel with the BrdU-pulsed samples in A, MK cells were collected at the indicated times and separated into total cell lysates (TCE), or fractionated into nucleosolic/cytosolic detergent-soluble extracts (S1) or chromatin-bound detergent-resistant extracts (P3). Immunoblotting with the indicated antibodies was performed on lysates from equal cell numbers loaded into each lane. The G₁-S transition in MK cells (12 hrs after release) is overlayed in gray.</p

<i>De novo</i> mRNA synthesis is not required in the final 3–4 hrs of G₁ for entry into S-phase.

<p>(<i>A</i>) Diagram illustrating the experimental design for the data obtained in <i>B</i>. (<i>B</i>) Balb/MK cells were synchronized in G₀ by EGF deprivation and then released into the cell cycle by re-addition of EGF. At the times indicated, control cells were pulsed with BrdU to determine the kinetics of progression through G₁ into S-phase (gray columns on left). Cells treated with 50 µM DRB at the times indicated were allowed to progress to the peak of S-phase at 15 hrs, at which time they were pulsed with BrdU to determine the percentage of cells that could enter S-phase following different times of DRB exposure (black columns on right). As a control, the DMSO carrier was added to a parallel culture at 1 hr and remained until the BrdU pulse at 15 hrs (white column on right). The 15 hr untreated control (gray column on right) indicates the maximum number of BrdU-labeled cells obtained. The means of triplicate counts of ∼200 cells/field+/−1 s. d. are shown. <i>C</i>) Asynchronous, logarithmically growing human MCF7 cells were treated with 50 µM DRB from time zero (squares), or not treated (circles), during a 24 hr period. BrdU was added at the beginning of the experiment and remained throughout the 24 hr period. At the indicated times, samples were fixed and processed for BrdU incorporation to determine the percentage of cells that had entered S-phase. The first time point was exposed to BrdU for 30 min before fixation. The means of triplicate counts of ∼250 cells/field+/−1 s. d. are shown.</p