HDAC6/aggresome processing pathway importance for inflammasome formation is context-dependent.

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1ß and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocyte cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context-dependent.

Expression and Crystallization of HDAC6 Tandem Catalytic Domains.

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.

Immunoprecipitation of HDAC6 and Interacting Proteins.

The lysine deacetylase HDAC6 has unique structural and functional properties: It contains tandem catalytic domains that can deacetylate a variety of proteins and a zinc finger domain that binds ubiquitin. HDAC6 has been implicated in a variety of biological processes, normal or pathological, such as cellular motility, stress response, cancer, neurodegeneration, or viral infection. Due to this, HDAC6 is considered an attractive therapeutic target, and there is a major interest to identify small molecule inhibitors. To gain a mechanistic understanding of how HDAC6 impacts these different biological processes, there is a continued need to discover additional substrates as well as interacting proteins in different paradigms. One approach to achieve this is to perform HDAC6 immunoprecipitations to identify partner proteins. We describe here our optimized protocols to immunoprecipitate HDAC6 with the goal to identify or validate interacting proteins.

TRIM25 Binds RNA to Modulate Cellular Anti-viral Defense.

TRIM25 is a multi-domain, RING-type E3 ubiquitin ligase of the tripartite motif family that has important roles in multiple RNA-dependent processes. In particular, TRIM25 functions as an effector of RIG-I and ZAP, which are innate immune sensors that recognize viral RNA and induce ubiquitin-dependent anti-viral response mechanisms. TRIM25 is reported to also bind RNA, but the molecular details of this interaction or its relevance to anti-viral defense have not been elucidated. Here, we characterize the RNA-binding activity of TRIM25 and find that the protein binds both single-stranded and double-stranded RNA. Multiple regions of TRIM25 contribute to this functionality, including the C-terminal SPRY domain and a lysine-rich motif in the linker segment connecting the SPRY and coiled-coil domains. RNA binding modulates TRIM25's ubiquitination activity in vitro, its localization in cells, and its anti-viral activity. Taken together with other studies, our results indicate that RNA binding by TRIM25 has at least three important functional consequences: by enhancing ubiquitination activity, either through allosteric effects or through clustering of multiple TRIM25 molecules; by modulating the multi-domain structure of the TRIM25 dimer, and thereby structural coupling of the SPRY and RBCC elements during the ubiquitination reaction; and by facilitating subcellular localization of the E3 ligase during virus infection.

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2-ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway.

Antiviral response pathways induce interferon by higher-order assembly of signaling complexes called signalosomes. Assembly of the RIG-I signalosome is regulated by K63-linked polyubiquitin chains, which are synthesized by the E3 ubiquitin ligase, TRIM25. We have previously shown that the TRIM25 coiled-coil domain is a stable, antiparallel dimer that positions two catalytic RING domains on opposite ends of an elongated rod. We now show that the RING domain is a separate self-association motif that engages ubiquitin-conjugated E2 enzymes as a dimer. RING dimerization is required for catalysis, TRIM25-mediated RIG-I ubiquitination, interferon induction, and antiviral activity. We also provide evidence that RING dimerization and E3 ligase activity are promoted by binding of the TRIM25 SPRY domain to the RIG-I effector domain. These results indicate that TRIM25 actively participates in higher-order assembly of the RIG-I signalosome and helps to fine-tune the efficiency of the RIG-I-mediated antiviral response.

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer.

Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication.