C. elegans Brat homologs regulate PAR protein-dependent polarity and asymmetric cell division

The evolutionary conserved PAR proteins control polarization and asymmetric division in many organisms. Recent work in Caenorhabditis elegans demonstrated that nos-3 and fbf-1/2 can suppress par-2(it5ts) lethality, suggesting that they participate in cell polarity by regulating the function of the anterior PAR-3/PAR-6/PKC-3 proteins. In Drosophila embryos, Nanos and Pumilio are homologous to NOS-3 and FBF-1/2 respectively and control cell polarity by forming a complex with the tumor suppressor Brat to inhibit Hunchback mRNA translation. In this study, we investigated the possibility that Brat could control cell polarity and asymmetric cell division in C. elegans. We found that disrupting four of the five C. elegans Brat homologs (Cebrats) individually results in suppression of par-2(it5ts) lethality, indicating that these genes are involved in embryonic polarity. Two of the Cebrats, ncl-1 and nhl-2, partially restore the localization of PAR proteins at the cortex. While mutations in the four Cebrat genes do not severely impair polarity, they display polarity-associated defects. Surprisingly, these defects are absent from nos-3 mutants. Similarly, while nos-3 controls PAR-6 protein levels, this is not the case for any of the Cebrats. Our results, together with results from Drosophila, indicate that Brat family members function in generating cellular asymmetries and suggest that, in contrast to Drosophila embryos, the C. elegans homologs of Brat and Nanos could participate in embryonic polarity via distinct mechanisms.     

Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.     

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.     

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.     

Characterization and biological function analysis of the trim3a gene from zebrafish (Danio rerio)

The biological significance of tripartite motif (TRIM) proteins is increasingly being appreciated due to their roles in a broad range of biological processes that associated with innate immunity. In this study, we have described the structural and functional analysis of TRIM3a from zebrafish. Annotation of domain architectures found that the TRIM3a fulfills the TRIM-NHL rule of domain composition with a Filamin/ABP280 domain and NHL repeats at its C-terminal region. In addition, the mRNA expression level of TRIM3a was the highest in brain, and with a relatively higher level in spleen, liver, and gill. A strong expression starting at 36 h post fertilization (hpf) was observed by real-time PCR and could be detected in brain by in situ hybridization, suggesting that TRIM3a protein might play an important role in brain development in zebrafish. Considering that TRIM3a has a RING finger domain, we expressed and purified the TRIM3a protein and performed ubiquitylation assays, our results showed that TRIM3a underwent self-polyubiquitylation in combination with E1, UbcH5c, biotin-ubiquitin in vitro. Meanwhile, TRIM3a-R without the RING domain was expressed and purified as well, in vitro ubiquitylation assays showed that the self-ubiquitylation of TRIM3a was dependent on its RING domain, suggesting that TRIM3a might function as a RING finger E3 ubiquitin ligase.     

In Silico Analysis of Functional Single Nucleotide Polymorphisms in the Human TRIM22 Gene

Tripartite motif protein 22 (TRIM22) is an evolutionarily ancient protein that plays an integral role in the host innate immune response to viruses. The antiviral TRIM22 protein has been shown to inhibit the replication of a number of viruses, including HIV-1, hepatitis B, and influenza A. TRIM22 expression has also been associated with multiple sclerosis, cancer, and autoimmune disease. In this study, multiple in silico computational methods were used to identify non-synonymous or amino acid-changing SNPs (nsSNP) that are deleterious to TRIM22 structure and/or function. A sequence homology-based approach was adopted for screening nsSNPs in TRIM22, including six different in silico prediction algorithms and evolutionary conservation data from the ConSurf web server. In total, 14 high-risk nsSNPs were identified in TRIM22, most of which are located in a protein interaction module called the B30.2 domain. Additionally, 9 of the top high-risk nsSNPs altered the putative structure of TRIM22''s B30.2 domain, particularly in the surface-exposed v2 and v3 regions. These same regions are critical for retroviral restriction by the closely-related TRIM5α protein. A number of putative structural and functional residues, including several sites that undergo post-translational modification, were also identified in TRIM22. This study is the first extensive in silico analysis of the highly polymorphic TRIM22 gene and will be a valuable resource for future targeted mechanistic and population-based studies.     

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 RNA binding of the mouse Pumilio-2 Puf domain

Puf proteins control translation through the interaction of a C-terminal Puf domain with specific sequences present in the 3′ untranslated region of messenger RNAs. In Drosophila, binding of the protein Pumilio to mRNA leads to translational repression which is required for anterior/posterior patterning during embryogenesis. The vertebrate Pumilio homologue 2 (Pum2) has been implicated in controlling germ cell development through interactions with the RNA binding proteins deleted in azoospermia (DAZ), DAZ-like (DAZL) and BOULE. We present the 1.6 Å resolution X-ray crystal structure of the Puf domain from murine Pum2 and demonstrate that this domain is capable of binding with nanomolar affinity to RNA sequences from the hunchback Nanos response element (NRE) and a previously identified Pum2 binding element (PBE).     

Biochemical and biophysical characterization of a chimeric TRIM21-TRIM5alpha protein

The tripartite motif (TRIM) protein, TRIM5α, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5α promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5α protein (TRIM5_Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5_Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5_Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5_Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5_Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.     

Negative regulation of diminutive cancer regulator through differentiation and microRNA pathway components in Drosophila cells

Drosophila model is intensively studied for the development of cancer. The diminutive (dMyc), a homolog of the human MYC gene, is responsible for cell- apoptosis and its upregulation is responsible for determining the fate of cancerous growth in humans and Drosophila model. This work implores the requirement of dMyc and its expression as one of the major regulator of cancer with other proteins and repression of dMyc mRNA in Drosophila S2 cells. Here we report protein complex of Argonaute 1 (AGO1), Bag of marbles (Bam), and Brain tumor (Brat) proteins and not the individual factor of this complex repression of dMyc mRNA in Drosophila Schneider 2 cells and promote differentiation in cystoblast of Drosophila ovary. These results exhibit the significant role of this complex, including master differentiation factor Bam with other various differentiation factor Brat and microRNA pathway component AGO1, which may negatively regulate dMyc mRNA and so the dMyc protein.     

Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function,but not Subunit Interaction of,the ADPR-Activated TRPM2 Channel

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca²⁺-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile⁶⁵⁸ to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.     

The role of Drosophila hyperplastic discs gene in spermatogenesis

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [ Martin et al. (1977) Dev Biol 55 , 213–232; Mansfield et al. (1994) Dev Biol 165 , 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [ Dorogova et al. (2008) BMC Cell Biol 9 , 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)‐binding protein C‐terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.     

Different subcellular localisations of TRIM22 suggest species-specific function

The B30.2/SPRY domain is present in many proteins, including various members of the tripartite motif (TRIM) protein family such as TRIM5α, which mediates innate intracellular resistance to retroviruses in several primate species. This resistance is dependent on the integrity of the B30.2 domain that evolves rapidly in primates and exhibits species-specific anti-viral activity. TRIM22 is another positively selected TRIM gene. Particularly, the B30.2 domain shows rapid evolution in the primate lineage and recently published data indicate an anti-viral function of TRIM22. We show here that human and rhesus TRIM22 localise to different subcellular compartments and that this difference can be assigned to the positively selected B30.2 domain. Moreover, we could demonstrate that amino acid changes in two variable loops (VL1 and VL3) are responsible for the different subcellular localisations.