Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. Here we report the results of a moderate-scale sequencing study aimed at identifying new genes contributing to predisposition for ALS. We performed whole exome sequencing of 2,874 ALS patients and compared them to 6,405 controls. Several known ALS genes were found to be associated, and the non-canonical IκB kinase family TANK-Binding Kinase 1 (TBK1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

Highlights d Developed 13 C-infusion method for studying T cell metabolism in vivo d T cell glucose use and bioenergetics differ between cell culture and mouse models d Glucose metabolism in T cells changes dynamically over an immune response d Glucose-dependent serine biosynthesis supports T cell proliferation in vivo

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.T he human papillomaviruses (HPVs) comprise more than 120 different virus types, each with a double-stranded DNA genome of approximately 8 kb. The HPVs have similar genome organizations, with regulatory functions encoded by the early (E) genes and structural components encoded by the late (L) genes (reviewed in reference 24). HPVs of different types differ in DNA sequences by 10% or more in the L1 gene, and the other viral genes exhibit a greater degree of sequence diversity between types (6, 15). Papillomaviruses are grouped into genera based on their L1 gene sequences and are further subdivided into species, with most of the HPVs in genus alpha or genus beta. The genus alpha HPVs infect the mucosal epithelium, and those that are the etiological agent responsible for the development of anogenital cancers (including cervical cancer) fall into genus alpha, species 7, and genus alpha, species 9. Beta-type HPVs infect the cutaneous epithelium.The HPV E6 protein has long been appreciated as a critical regulator of the viral life cycle and driver of tumorigenesis for the high-risk HPVs. Through the action of the HPV E7 protein, the G 1 -S checkpoint is bypassed in infected cells by the inactivation of the retinoblastoma tumor suppressor protein (pRB1) (17,18,46). This results in a cellular environment that is conducive to the replication of the viral DNA but in which proapoptotic signals have been triggered. One crucial function of high-risk HPV E6 proteins is to counteract the effects of p53 following this trigger, and this is accomplished by the targeted ubiquitylati...

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.T he many types of human papillomaviruses (HPVs) that have been described exhibit considerable diversity. The HPVs are DNA viruses with a tropism specific for squamous epithelial cells. More than 120 HPVs have been identified and cloned to date, and these share a conserved genomic structure with eight to 10 ORFs encoded on one strand of a small double-stranded circular DNA genome (1). The ORFs involved in fundamental processes such as DNA replication or capsid formation are well conserved. Other ORFs, such as E6 and E7, have some conserved features but are more divergent at the nucleotide and protein level. Consistent with these differences, the lesions that are caused by infection with different HPVs and the propensity for these lesions to progress to cancer vary as well (2). A subset of the HPVs are the primary etiological agent in the development of cervical cancer, and other HPVs cause genital or cutaneous warts or other skin lesions. Relatively little is known about how these sequence differences translate into different biological outcomes in infected human cells. Thus, there exists an opportunity to systematically define features of diverse HPVs and to understand at the molecular level how their varied genetic compositions result in different disease states.The standard phylogeny of the HPVs is based on the sequence of the L1 gene, and a virus with an L1 DNA sequence that differs by 10% or more from other HPV L1s is designated as a separate type (1). Similar HPV types are grouped into species and further into genera. The majority of the HPVs identi...