Autophagy, the degradation of cytoplasmic components, is an evolutionarily conserved homeostatic process involved in environmental adaptation, lifespan determination and tumour development. The tumor suppressor Beclin1 is part of the PI(3) kinase class III (PI(3)KC3) lipid-kinase complex that induces autophagy. The autophagic activity of the Beclin1-PI(3)KC3 complex, however, is suppressed by Bcl-2. Here, we report the identification of a novel coiled-coil UV irradiation resistance-associated gene (UVRAG) as a positive regulator of the Beclin1-PI(3)KC3 complex. UVRAG, a tumour suppressor candidate that is monoallelically mutated at high frequency in human colon cancers, associates with the Beclin1-Bcl-2-PI(3)KC3 multiprotein complex, where UVRAG and Beclin1 interdependently induce autophagy. UVRAG-mediated activation of the Beclin1-PI(3)KC3 complex promotes autophagy and also suppresses the proliferation and tumorigenicity of human colon cancer cells. These results identify UVRAG as an essential component of the Beclin1-PI(3)KC3 lipid kinase complex that is an important signalling checkpoint for autophagy and tumour-cell growth.

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X L , MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.

Condensins are key mediators of chromosome condensation across organisms. Like other condensins, the bacterial MukBEF condensin complex consists of an SMC family protein dimer containing two ATPase head domains, MukB, and two interacting subunits, MukE and MukF. We report complete structural views of the intersubunit interactions of this condensin along with ensuing studies that reveal a role for the ATPase activity of MukB. MukE and MukF together form an elongated dimeric frame, and MukF's C-terminal winged-helix domains (C-WHDs) bind MukB heads to constitute closed ring-like structures. Surprisingly, one of the two bound C-WHDs is forced to detach upon ATP-mediated engagement of MukB heads. This detachment reaction depends on the linker segment preceding the C-WHD, and mutations on the linker restrict cell growth. Thus ATP-dependent transient disruption of the MukB-MukF interaction, which creates openings in condensin ring structures, is likely to be a critical feature of the functional mechanism of condensins.

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine γ-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.

Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

SUMMARY Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism towards glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an “actionable” therapeutic target in cancer.

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.