Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

Lre1 directly inhibits the NDR/Lats kinase Cbk1 at the cell division site in a phosphorylation-dependent manner.

BACKGROUND: The nuclear Dbf2 related (NDR) family of protein kinases play important roles in cell-cycle regulation, apoptosis, cell morphogenesis, and development in a variety of organisms. In budding yeast, the NDR kinase complex composed of Cbk1 and its regulatory subunit, Mob2, have an established role in the control of cell separation/abscission that follows cytokinesis. Whereas the activators of Cbk1-Mob2 have been more extensively described, the mechanisms that restrict or inhibit Cbk1-Mob2 catalytic activity remain largely unknown. RESULTS: We identified the protein Lre1 as a direct inhibitor of Cbk1-Mob2 catalytic activity. We show that Lre1 accumulates at the cell division site in late anaphase and associates with both Mob2 and Cbk1 in vivo and in vitro. Biochemical and functional analysis established that the ability of Lre1 to associate with Cbk1-Mob2 was reduced by mitotic Cdk1 activity and promoted by Cdc14 phosphatase at the end of mitosis. The inhibition of Cbk1-Mob2 by Lre1 was critical to promote the survival of cells lacking the actomyosin driven pathway of cytokinesis. CONCLUSIONS: We established Lre1 as a direct inhibitor of the NDR kinase Cbk1-Mob2, which is regulated in a cell-cycle-dependent manner. We propose that similar inhibitory proteins may also provide fine tuning for the activity of NDR kinases in other organisms.

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis.

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.

Elm1 kinase activates the spindle position checkpoint kinase Kin4.

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

Protein NO52--a constitutive nucleolar component sharing high sequence homologies to protein NO66.

The nucleolus is the most prominent intranuclear structure of almost all protein-synthesizing cells. It compromises a well-defined functional compartmentalization and a high complexity of molecular constituents. Here, we report on the identification and molecular characterization of a novel constitutive nucleolar component--protein NO52--that is present in diverse species from Xenopus laevis to human. The cDNA-deduced amino acid sequence of protein NO52 defines a polypeptide of a calculated mass of 52.8 kDa and an isoelectric point of 6.7. Inspection of the primary sequence disclosed that the protein contains a JmjC domain and is highly sequence-related to the recently described nucleolar protein NO66. Immunolocalization studies revealed that protein NO52 is highly concentrated in the granular component of nucleoli and this characteristic intranuclear distribution is significantly affected by treatment of cells with (i) RNase A, (ii) actinomycin D and (iii) serum starvation. Interestingly, protein NO52 has been identified as a constituent of free preribosomal particles but is absent from cytoplasmic ribosomes. Analyses of immunocomplexes isolated from cellular extracts with an NO52-specific antibody by MALDI mass spectrometry further confirmed the interaction of protein NO52 with various ribosomal proteins as well as with a distinct set of non-ribosomal nucleolar proteins. The dependence of the nucleolar accumulation of the protein on ongoing rRNA transcription and the cellular metabolic state strongly suggest that protein NO52 is directly involved in ribosome biogenesis, most likely during the assembly process of preribosomal particles.