Elucidating the Antiviral Mechanism of Different MARCH Factors.

The membrane-associated RING-CH (MARCH) proteins belong to a family of E3 ubiquitin ligases, whose main function is to remove transmembrane proteins from the plasma membrane. Recent work has shown that the human MARCH1, 2, and 8 are antiretroviral factors that target the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins by reducing their incorporation in the budding virions. Nevertheless, the dearth of information regarding the antiviral mechanism of this family of proteins necessitates further examination. In this study, using both the human MARCH proteins and their mouse homologues, we provide a comprehensive analysis of the antiretroviral mechanism of this family of proteins. Moreover, we show that human MARCH proteins restrict to various degrees the envelope glycoproteins of a diverse number of viruses. This report sheds light on the important antiviral function of MARCH proteins and their significance in cell intrinsic immunity.IMPORTANCE This study examines the mechanism utilized by different MARCH proteins to restrict retrovirus infection. MARCH proteins block the incorporation of envelope glycoproteins to the budding virions. In this report, by comparing the human and mouse MARCH genes and using murine leukemia virus (MLV) and HIV-1, we identify differences in the mechanism of restriction among MARCH proteins. Furthermore, we perform a comprehensive analysis on a number of envelope glycoproteins and show that MARCH proteins have broad antiviral functions.

Substrate recognition by TRIM and TRIM-like proteins in innate immunity.

TRIM (Tripartite motif) and TRIM-like proteins have emerged as an important class of E3 ligases in innate immunity. Their functions range from activation or regulation of innate immune signaling pathway to direct detection and restriction of pathogens. Despite the importance, molecular mechanisms for many TRIM/TRIM-like proteins remain poorly characterized, in part due to challenges of identifying their substrates. In this review, we discuss several TRIM/TRIM-like proteins in RNA sensing pathways and viral restriction functions. We focus on those containing PRY-SPRY, the domain most frequently used for substrate recognition, and discuss emerging mechanisms that are commonly utilized by several TRIM/TRIM-like proteins to tightly control their interaction with the substrates.

Emerging RNA-binding roles in the TRIM family of ubiquitin ligases.

TRIM proteins constitute a large, diverse and ancient protein family which play a key role in processes including cellular differentiation, autophagy, apoptosis, DNA repair, and tumour suppression. Mostly known and studied through the lens of their ubiquitination activity as E3 ligases, it has recently emerged that many of these proteins are involved in direct RNA binding through their NHL or PRY/SPRY domains. We summarise the current knowledge concerning the mechanism of RNA binding by TRIM proteins and its biological role. We discuss how RNA-binding relates to their previously described functions such as E3 ubiquitin ligase activity, and we will consider the potential role of enrichment in membrane-less organelles.

Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway.

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

iUUCD 2.0: an update with rich annotations for ubiquitin and ubiquitin-like conjugations.

Here, we described the updated database iUUCD 2.0 (http://iuucd.biocuckoo.org/) for ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin-protein ligases (E3s), deubiquitinating enzymes (DUBs), ubiquitin/ubiquitin-like binding domains (UBDs) and ubiquitin-like domains (ULDs), which act as key regulators in modulating ubiquitin and ubiquitin-like (UB/UBL) conjugations. In total, iUUCD 2.0 contained 136 512 UB/UBL regulators, including 1230 E1s, 5636 E2s, 93 343 E3s, 9548 DUBs, 30 173 UBDs and 11 099 ULDs in 148 eukaryotic species. In particular, we provided rich annotations for regulators of eight model organisms, especially in humans, by compiling and integrating the knowledge from nearly 70 widely used public databases that cover cancer mutations, single nucleotide polymorphisms (SNPs), mRNA expression, DNA and RNA elements, protein-protein interactions, protein 3D structures, disease-associated information, drug-target relations, post-translational modifications, DNA methylation and protein expression/proteomics. Compared with our previously developed UUCD 1.0 (∼0.41 GB), iUUCD 2.0 has a size of ∼32.1 GB of data with a >75-fold increase in data volume. We anticipate that iUUCD 2.0 can be a more useful resource for further study of UB/UBL conjugations.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

BACKGROUND: The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. RESULTS: Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. CONCLUSION: Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.

RING-, HECT-, and RBR-type E3 Ubiquitin Ligases: Involvement in Human Cancer.

In the ubiquitylation system, E3 ubiquitin ligases play a key role in determining substrate specificity and catalyzing the transfer of ubiquitin from E2 enzymes to the substrate. Growing evidence has shown that E3 ubiquitin ligases are involved in cancer development and progression. The RING-type and HECT-type E3 ligases are the classically categorized groups of E3 ubiquitin ligases, and more of these enzymes are being shown to be potential targets for cancer therapy. The recently classified RBR E3 ligases catalyze the transfer of ubiquitin by a RING/HECT hybrid-like mechanism. Notably, these ligases are also emphasized as important potential candidates for targets of cancer treatment drugs. The present review provides an overview of the RING-, HECT- and RBR-type E3 ligases, and discusses their roles in cancer and cancer therapy.

Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.

Efficient and regular performance of Ubiquitin Proteasome System and Autophagy continuously eliminate deleterious accumulation of nonnative protiens. In cellular quality control system, E3 ubiquitin ligases are significant employees for defense mechanism against abnormal toxic proteins. Few findings indicate that lack of functions of E3 ubiquitin ligases can be a causative factor of neurodevelopmental disorders, neurodegeneration, cancer and ageing. However, the detailed molecular pathomechanism implying E3 ubiquitin ligases in cellular functions in multifactorial disease conditions are not well understood. This article systematically represents the unique characteristics, molecular nature, and recent developments in the knowledge of neurobiological functions of few crucial E3 ubiquitin ligases. Here, we review recent literature on the roles of E6-AP, HRD1 and ITCH E3 ubiquitin ligases in the neuro-pathobiological mechanisms, with precise focus on the processes of neurodegeneration, and thereby propose new lines of potential targets for therapeutic interventions.

Origin and diversification of TRIM ubiquitin ligases.

Most proteins of the TRIM family (also known as RBCC family) are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Ancient origin of animal U-box ubiquitin ligases.

BACKGROUND: The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described. RESULTS: Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans (UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4) were already present in the ancestor of all current metazoans and the seventh (WDSUB1) is found in placozoans, cnidarians and bilaterians. The fact that only 4-5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families (RBR, HECT) of ubiquitin ligases. CONCLUSIONS: Finding that the patterns of emergence, diversification and loss of three unrelated families of ubiquitin ligases (RBR, HECT and U-box) are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system.

The genetics of Parkinson disease.

Parkinson disease (PD) is the second most common neurodegenerative disorder. In most instances, PD is thought to result from a complex interaction between multiple genetic and environmental factors, though rare monogenic forms of the disease do exist. Mutations in 6 genes (SNCA, LRRK2, PRKN, DJ1, PINK1, and ATP13A2) have conclusively been shown to cause familial parkinsonism. In addition, common variation in 3 genes (MAPT, LRRK2, and SNCA) and loss-of-function mutations in GBA have been well-validated as susceptibility factors for PD. The function of these genes and their contribution to PD pathogenesis remain to be fully elucidated. The prevalence, incidence, clinical manifestations, and genetic components of PD are discussed in this review.

Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions.

The Nedd4 (neural precursor cell-expressed developmentally downregulated gene 4) family of ubiquitin ligases (E3s) is characterized by a distinct modular domain architecture, with each member consisting of a C2 domain, 2-4 WW domains, and a HECT-type ligase domain. Of the nine mammalian members of this family, Nedd4 and its close relative, Nedd4-2, represent the ancestral ligases with strong similarity to the yeast, Rsp5. In Saccharomyces cerevisiae Rsp5 has a key role in regulating the trafficking, sorting, and degradation of a large number of proteins in multiple cellular compartments. However, in mammals the Nedd4 family members, including Nedd4 and Nedd4-2, appear to have distinct functions, thereby suggesting that these E3s target specific proteins for ubiquitylation. In this article we focus on the biology and emerging functions of Nedd4 and Nedd4-2, and review recent in vivo studies on these E3s.

Evolutionary origin of the tumor suppressor hyperplastic discs protein.

Previous evolutionary study of the tumor suppressor Merlin revealed that this protein family was produced by very early metazoans with the exception of some or all flatworm lineages [Golovnina et al., 2005]. We ask whether other tumor-suppressor proteins had also been in existence in these times and focus our attention on hyperplastic discs (Hyd) protein, a classic tumor suppressor in Drosophila melanogaster which, when mutated, may cause over-proliferation and malignancy. Phylogenetic analysis of the Hyd protein indicates that it was present among metazoa by the time Trichoplax adhaerens had emerged from the common unicellular ancestor of the Animalia.

IMP modulates KSR1-dependent multivalent complex formation to specify ERK1/2 pathway activation and response thresholds.

The Ras effector and ubiquitin-protein isopeptide ligase family member IMP acts as a steady-state resistor within the Raf-MEK-ERK kinase module. IMP concentrations are regulated by Ras through induction of autodegradation and can modulate signal/response thresholds by directly limiting the assembly of functional KSR1-dependent Raf.MEK complexes. Here, we show that the capacity of IMP to inhibit signal propagation through Raf to MEK is a consequence of disrupting KSR1 homooligomerization and B-Raf/c-Raf hetero-oligomerization. This impairs both the recruitment of MEK to activated Raf family members and the contribution of Raf oligomers to c-Raf kinase activation. Our observations indicate that human KSR1 proteins promote assembly of multivalent Raf.MEK complexes that are required for c-Raf kinase activation and functional coupling of active kinases to downstream substrates. This property is engaged by IMP for modulation of signal amplitude.

Ubiquitin ligases in cancer: ushers for degradation.

The regulated degradation of cellular proteins by the ubiquitin-proteasome system impacts a range of vital cellular processes in both normal and cancerous cells. An ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) catalyzes the conjugation of the protein ubiquitin to a target protein and, thereby, tags that protein for recognition and destruction by the proteasome. Ubiquitin ligases are particularly interesting because they determine substrate selection. This review examines the role of dysregulated ubiquitin ligase activity in the development and progression of various cancers, and highlights why ubiquitin ligases have emerged as extremely attractive targets for therapeutic intervention in a number of human malignancies.

Genetic and expression aberrations of E3 ubiquitin ligases in human breast cancer.

Recent studies revealed that E3 ubiquitin ligases play important roles in breast carcinogenesis. Clinical research studies have found that (epi)-genetic (deletion, amplification, mutation, and promoter methylation) and expression aberration of E3s are frequent in human breast cancer. Furthermore, many studies have suggested that many E3s are either oncogenes or tumor suppressor genes in breast cancer. In this review, we provide a comprehensive summary of E3s, which have genetic and/or expression aberration in breast cancer. Most cancer-related E3s regulate the cell cycle, p53, transcription, DNA repair, cell signaling, or apoptosis. An understanding of the oncogenic potential of the E3s may facilitate identifying and developing individual E3s as diagnosis markers and drug targets in breast cancer.