Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Reduced Levels of Lagging Strand Polymerases Shape Stem Cell Chromatin.

Stem cells display asymmetric histone inheritance while non-stem progenitor cells exhibit symmetric patterns in the Drosophila male germline lineage. Here, we report that components involved in lagging strand synthesis, such as DNA polymerase α and δ (Polα and Polδ), have significantly reduced levels in stem cells compared to progenitor cells. Compromising Polα genetically induces the replication-coupled histone incorporation pattern in progenitor cells to be indistinguishable from that in stem cells, which can be recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, stem cell-derived chromatin fibers display a higher degree of old histone recycling by the leading strand compared to progenitor cell-derived chromatin fibers. However, upon reducing Polα levels in progenitor cells, the chromatin fibers now display asymmetric old histone recycling just like GSC-derived fibers. The old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S-phase and M-phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in non-stem cells in a manner similar to that in stem cells.

Skp1 proteins are structural components of the synaptonemal complex in C. elegans.

The synaptonemal complex (SC) is a zipper-like protein assembly that links homologous chromosomes to regulate recombination and segregation during meiosis. The SC has been notoriously refractory to in vitro reconstitution, thus leaving its molecular organization largely unknown. Here, we report a moonlighting function of two paralogous S-phase kinase-associated protein 1 (Skp1)-related proteins (SKR-1 and SKR-2), well-known adaptors of the Skp1-Cul1-F-box (SCF) ubiquitin ligase, as the key missing components of the SC in Caenorhabditis elegans. SKR proteins repurpose their SCF-forming interfaces to dimerize and interact with meiosis-specific SC proteins, thereby driving synapsis independent of SCF activity. SKR-1 enables the formation of the long-sought-after soluble complex with previously identified SC proteins in vitro, which we propose it to represent a complete SC building block. Our findings demonstrate how a conserved cell cycle regulator has been co-opted to interact with rapidly evolving meiotic proteins to construct the SC and provide a foundation for understanding its structure and assembly mechanisms.

Release of CHK-2 from PPM-1.D anchorage schedules meiotic entry.

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans. Interfering with all three branches causes tumorous germ lines. SCFPROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCFPROM-1-mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.