Genetic and pathogenic characterizations of a naturally occurring reassortant and homologous recombinant strain of the classical infectious bursal disease virus re-emerging in chickens in southern China.

Infectious bursal disease (IBD) classical virus strain (cIBDV) can cause morbidity and mortality in young chickens with severe long-term immunosuppression. However, since the emergence and widespread prevalence of very virulent strain (vvIBDV) in China from 1991, reports of cIBDV have become rare. A novel reassortant and recombinant strain GXYL211225 (genotype A1aB1a) with segment A originating from the classical strain (A1a) and segment B from the attenuated vaccine strain (B1a) was characterized in the study. Notably, segment A resulted from recombination between the cIBDV strains 150127-0.2 and Faragher52-70, expressing as a backbone from 150127-0.2, where a fragment located at the position of nucleotide (nt) 519-1 410 was replaced by the corresponding region of Faragher52-70. The infection of GXYL211225 caused mortality in SPF chicken embryos, despite lacking the critical amino acid (aa) residues 253H, 279 N and 284A associated with the cellular tropism, and induced significant cytopathic effect (CPE) on a wide range of cells, confirming its natural cell-adapted character. Furthermore, the challenge experiment of GXYL211225 was performed on the commercial Three-yellow chickens of 4-week-old, and with the vvIBDV HLJ-0504-like strain NN1172 and the novel variant (nv) IBDV strain QZ191002 as the comparison. All the challenged birds experienced reduced body-weight gain. QZ191002 infected birds showed no obvious clinical symptoms or mortality, while those of NN1172 and GXYL211225 showed typical IBD symptoms and resulted in 20% (2/10) and 10% (1/10) of mortality rates, respectively. At 7 days post-challenge (dpc), the damages of bursal of Fabricius (BF) varied among groups, with NN1172 causing the most severe lesions, followed by GXYL211225, and then QZ191002. It was also found that the pathogenicity was correlated positively with the viral load, aligning with the histopathological severity in BF. The study confirms the rapid and diverse evolution of the re-emerged classical strains in the field and emphasizes the need to monitor the changes of IBDV on both the genetic and pathogenic aspects for the effective control of the disease.

Discovery of a cooperative mode of inhibiting RIPK1 kinase.

RIPK1, a death domain-containing kinase, has been recognized as an important therapeutic target for inhibiting apoptosis, necroptosis, and inflammation under pathological conditions. RIPK1 kinase inhibitors have been advanced into clinical studies for the treatment of various human diseases. One of the current bottlenecks in developing RIPK1 inhibitors is to discover new approaches to inhibit this kinase as only limited chemotypes have been developed. Here we describe Necrostatin-34 (Nec-34), a small molecule that inhibits RIPK1 kinase with a mechanism distinct from known RIPK1 inhibitors such as Nec-1s. Mechanistic studies suggest that Nec-34 stabilizes RIPK1 kinase in an inactive conformation by occupying a distinct binding pocket in the kinase domain. Furthermore, we show that Nec-34 series of compounds can synergize with Nec-1s to inhibit RIPK1 in vitro and in vivo. Thus, Nec-34 defines a new strategy to target RIPK1 kinase and provides a potential option of combinatorial therapy for RIPK1-mediated diseases.

Probing Protein-Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns.

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.

Structure of Myosin VI/Tom1 complex reveals a cargo recognition mode of Myosin VI for tethering.

Myosin VI plays crucial roles in diverse cellular processes. In autophagy, Myosin VI can facilitate the maturation of autophagosomes through interactions with Tom1 and the autophagy receptors, Optineurin, NDP52 and TAX1BP1. Here, we report the high-resolution crystal structure of the C-terminal cargo-binding domain (CBD) of Myosin VI in complex with Tom1, which elucidates the mechanistic basis underpinning the specific interaction between Myosin VI and Tom1, and uncovers that the C-terminal CBD of Myosin VI adopts a unique cargo recognition mode to interact with Tom1 for tethering. Furthermore, we show that Myosin VI can serve as a bridging adaptor to simultaneously interact with Tom1 and autophagy receptors through two distinct interfaces. In all, our findings provide mechanistic insights into the interactions of Myosin VI with Tom1 and relevant autophagy receptors, and are valuable for further understanding the functions of these proteins in autophagy and the cargo recognition modes of Myosin VI.

Mechanistic insights into the interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1.

NDP52 and TAX1BP1, two SKIP carboxyl homology (SKICH) domain-containing autophagy receptors, play crucial roles in selective autophagy. The autophagic functions of NDP52 and TAX1BP1 are regulated by TANK-binding kinase 1 (TBK1), which may associate with them through the adaptor NAP1. However, the molecular mechanism governing the interactions of NAP1 with NDP52 and TAX1BP1, as well as the effects induced by TBK1-mediated phosphorylation of NDP52 and TAX1BP1, remains elusive. Here, we report the atomic structures of the SKICH regions of NDP52 and TAX1BP1 in complex with NAP1, which not only uncover the mechanistic bases underpinning the specific interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1 but also reveal the binding mode of a SKICH domain. Moreover, we uncovered that the SKICH domains of NDP52 and TAX1BP1 share a general binding mode to interact with NAP1. Finally, we also evaluated the currently known TBK1-mediated phosphorylation sites in the SKICH domains of NDP52 and TAX1BP1 on the basis of their interactions with NAP1. In all, our findings provide mechanistic insights into the interactions of NAP1 with NDP52 and TAX1BP1, and are valuable for further understanding the functions of these proteins in selective autophagy.

Mechanistic Insights into Recognitions of Ubiquitin and Myosin VI by Autophagy Receptor TAX1BP1.

TAX1BP1, a ubiquitin-binding adaptor, plays critical roles in the innate immunity and selective autophagy. During autophagy, TAX1BP1 may not only function as an autophagy receptor to recruit ubiquitylated substrates for autophagic degradation, but also serve as a Myosin VI cargo adaptor protein for mediating the maturation of autophagosome. However, the mechanistic basis underlying the specific interactions of TAX1BP1 with ubiquitin and Myosin VI remains elusive. Here, using biochemical, NMR and structural analyses, we elucidate the detailed binding mechanism and uncover the key determinants for the interaction between TAX1BP1 and ubiquitin. In addition, we reveal that both tandem zinc-fingers of TAX1BP1 and the conformational rigidity between them are required for the Myosin VI binding of TAX1BP1, and ubiquitin and Myosin VI are mutually exclusive in binding to TAX1BP1. Collectively, our findings provide mechanistic insights into the dual functions of TAX1BP1 in selective autophagy.

Structural insights into the ubiquitin recognition by OPTN (optineurin) and its regulation by TBK1-mediated phosphorylation.

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.

Structural Insights into SHARPIN-Mediated Activation of HOIP for the Linear Ubiquitin Chain Assembly.

The linear ubiquitin chain assembly complex (LUBAC) is the sole identified E3 ligase complex that catalyzes the formation of linear ubiquitin chain, and it is composed of HOIP, HOIL-1L, and SHARPIN. The E3 activity of HOIP can be effectively activated by HOIL-1L or SHARPIN, deficiency of which leads to severe immune system disorders. However, the underlying mechanism governing the HOIP-SHARPIN interaction and the SHARPIN-mediated activation of HOIP remains elusive. Here, we biochemically and structurally demonstrate that the UBL domain of SHARPIN specifically binds to the UBA domain of HOIP and thereby associates with and activates HOIP. We further uncover that SHARPIN and HOIL-1L can separately or synergistically bind to distinct sites of HOIP UBA with induced allosteric effects and thereby facilitate the E2 loading of HOIP for its activation. Thus, our findings provide mechanistic insights into the assembly and activation of LUBAC.

Structural insights into the interaction and disease mechanism of neurodegenerative disease-associated optineurin and TBK1 proteins.

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients.

Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins.

FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.