C9orf72: At the intersection of lysosome cell biology and neurodegenerative disease.

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith-Magenis chromosome region 8 (SMCR8) and WD repeat-containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72-containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.

C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling.

Hexanucleotide expansion in an intron of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. However, beyond bioinformatics predictions that suggested structural similarity to folliculin, the Birt-Hogg-Dubé syndrome tumor suppressor, little is known about the normal functions of the C9orf72 protein. To address this problem, we used genome-editing strategies to investigate C9orf72 interactions, subcellular localization, and knockout (KO) phenotypes. We found that C9orf72 robustly interacts with SMCR8 (a protein of previously unknown function). We also observed that C9orf72 localizes to lysosomes and that such localization is negatively regulated by amino acid availability. Analysis of C9orf72 KO, SMCR8 KO, and double-KO cell lines revealed phenotypes that are consistent with a function for C9orf72 at lysosomes. These include abnormally swollen lysosomes in the absence of C9orf72 and impaired responses of mTORC1 signaling to changes in amino acid availability (a lysosome-dependent process) after depletion of either C9orf72 or SMCR8. Collectively these results identify strong physical and functional interactions between C9orf72 and SMCR8 and support a lysosomal site of action for this protein complex.

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration.

ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.

Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8-15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization.

Molecular basis of antiangiogenic thrombospondin-1 type 1 repeat domain interactions with CD36.

OBJECTIVE: Antiangiogenic activity of thrombospondin-1 and related proteins is mediated by interactions between thrombospondin type 1 repeat (TSR) domains and the CD36, LIMP-2, Emp sequence homology (CLESH) domain of the endothelial cell receptor CD36. We sought to characterize key molecular determinants of the interaction between thrombospondin-1 TSR domains and the CD36 CLESH domain. APPROACH AND RESULTS: Recombinant thrombospondin-1 TSR2 and TSR(2,3) constructs inhibited microvascular endothelial cell migration, microvascular endothelial cell tube formation, and vessel sprouting in aortic ring assays. Interaction with CD36 CLESH decoy peptides negated these effects. Mutational analyses identified a cluster of residues that confer positive charge to the TSR2 surface and mediate interaction with CD36 CLESH. Antiangiogenic activity was significantly reduced by charge-neutralizing mutations of the Arg-Trp ladder in TSR2, but not TSR3. Additionally, I438 and K464 of TSR2 were shown to be required for CD36 CLESH binding to TSR2. A complementary acidic cluster within CD36 CLESH is also required for antiangiogenic activity. CONCLUSIONS: Thrombospondin-1 interacts with CD36 CLESH through electrostatic interactions mediated by a positively charged TSR2 surface and multiple negatively charged CD36 CLESH residues. Two key residues serve as specificity determinants that identify other TSR domains that interact with CD36 CLESH.

Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates.

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.