A Comprehensive Assessment of Genetic and Epigenetic Alterations Identifies Frequent Variations Impacting Six Prototypic SCF Complex Members

The SKP1, CUL1, F-box protein (SCF) complex represents a family of 69 E3 ubiquitin ligases that poly-ubiquitinate protein substrates marking them for proteolytic degradation via the 26S proteasome. Established SCF complex targets include transcription factors, oncoproteins and tumor suppressors that modulate cell cycle activity and mitotic fidelity. Accordingly, genetic and epigenetic alterations involving SCF complex member genes are expected to adversely impact target regulation and contribute to disease etiology. To gain novel insight into cancer pathogenesis, we determined the prevalence of genetic and epigenetic alterations in six prototypic SCF complex member genes (SKP1, CUL1, RBX1, SKP2, FBXW7 and FBXO5) from patient datasets extracted from The Cancer Genome Atlas (TCGA). Collectively, ~45% of observed SCF complex member mutations are predicted to impact complex structure and/or function in 10 solid tumor types. In addition, the distribution of encoded alterations suggest SCF complex members may exhibit either tumor suppressor or oncogenic mutational profiles in a cancer type dependent manner. Further bioinformatic analyses reveal the potential functional implications of encoded alterations arising from missense mutations by examining predicted deleterious mutations with available crystal structures. The SCF complex also exhibits frequent copy number alterations in a variety of cancer types that generally correspond with mRNA expression levels. Finally, we note that SCF complex member genes are differentially methylated across cancer types, which may effectively phenocopy gene copy number alterations. Collectively, these data show that SCF complex member genes are frequently altered at the genetic and epigenetic levels in many cancer types, which will adversely impact the normal targeting and timely destruction of protein substrates, which may contribute to the development and progression of an extensive array of cancer types.

Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis

SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

The SCF Complex Is Essential to Maintain Genome and Chromosome Stability

The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.

Regulation of Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Expression by F-Box/LRR-Repeat (FBXL) Protein in Wheat (Triticum aestivum L.)

F-box proteins are substrate recognition components of the Skp1-Cullin-F-box (SCF) complex, which performs many important biological functions including the degradation of numerous proteins via the ubiquitin–26S proteasome system. In this study, we isolated the gene encoding the F-box/LRR-repeat (FBXL) protein from wheat (Triticum aestivum L.) seedlings and validated that the TaFBXL protein is a component of the SCF complex. Yeast two-hybrid assays revealed that TaFBXL interacts with the wheat glycosylphosphatidylinositol-anchored protein (TaGPI-AP). The green fluorescent protein (GFP) fusion protein of TaFBXL was detected in the nucleus and plasma membrane, whereas that of TaGPI-AP was observed in the cytosol and probably also plasma membrane. yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed that TaFBXL specifically interacts with TaGPI-AP in the nucleus and plasma membrane, and TaGPI-AP is targeted by TaFBXL for degradation via the 26S proteasome system. In addition, TaFBXL and TaGPI-AP showed antagonistic expression patterns upon treatment with indole-3-acetic acid (IAA), and the level of TaGPI-AP was higher in tobacco leaves treated with both MG132 (proteasome inhibitor) and IAA than in leaves treated with either MG132 or IAA. Taken together, our data suggest that TaFBXL regulates the TaGPI-AP protein level in response to exogenous auxin application.

Genome-Wide Analysis of Almond Skp1 Family Members And Identification of Self-Incompatibility-Related PsdSSK1

S-phase kinase-associated protein 1 ( Skp1 ) is a component of the SKP1-Cullin1-F-box (SCF) complex, which facilitates ubiquitin-mediated protein degradation in eukaryotes. SLF interacting Skp1-like 1 ( SSK1 ), which is located outside the S-site, is a newly discovered type of self-incompatibility-related pollen-specific gene whose product interacts with pollen determinants. In this study, we identified 18 members of the Prunus dulcis Skp1 (PsdSkp1) family that were unevenly distributed across six chromosomes of the almond genome. The phylogenetic tree revealed that the PsdSkp1 members could divided into four groups: I, II, III, and IV. The conserved motifs, conserved domains, and motif structures of the PsdSSK1 and PsdSkp1-12 proteins are similar. There were three pairs of fragment duplication genes and one pair of tandem repeat genes, and their functions were highly evolutionarily conserved. Transcriptome data showed that PsdSkp1 was expressed in almond flower tissues, that its expression was affected by freezing stress, and that its expression changed significantly in cross-pollination. Molecular experiments proved that PsdSSK1 and PsdSkp1-12 had the same protein sequence, and the genes were significantly highly expressed mainly in the pollen or throughout pollen cells, with subcellular localization in the cytoplasm. PsdSkp1-12 is an ssk1 gene in almond, and it may be involved in the SCF complex to regulate the almond pollen response via SI and cross-compatibility. In conclusion, our research lays a foundation for future functional research on PsdSkp1 members, especially for exploring the mechanism of almond SI.

Ubiquitination in Plant Meiosis: Recent Advances and High Throughput Methods

Meiosis is a specialized cell division which is essential to sexual reproduction. The success of this highly ordered process involves the timely activation, interaction, movement, and removal of many proteins. Ubiquitination is an extraordinarily diverse post-translational modification with a regulatory role in almost all cellular processes. During meiosis, ubiquitin localizes to chromatin and the expression of genes related to ubiquitination appears to be enhanced. This may be due to extensive protein turnover mediated by proteasomal degradation. However, degradation is not the only substrate fate conferred by ubiquitination which may also mediate, for example, the activation of key transcription factors. In plant meiosis, the specific roles of several components of the ubiquitination cascade—particularly SCF complex proteins, the APC/C, and HEI10—have been partially characterized indicating diverse roles in chromosome segregation, recombination, and synapsis. Nonetheless, these components remain comparatively poorly understood to their counterparts in other processes and in other eukaryotes. In this review, we present an overview of our understanding of the role of ubiquitination in plant meiosis, highlighting recent advances, remaining challenges, and high throughput methods which may be used to overcome them.

Host F-box protein 22 enhances the uptake of Brucella by macrophages and drives a sustained release of pro-inflammatory cytokines through degradation of the anti-inflammatory effector proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella -macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella -infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella , including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, β-TrCP in Brucella -macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

A WD40 repeat-like protein pathway connects F-BOX STRESS INDUCED (FBS) proteins to the NIGT1.1 transcriptional repressor in Arabidopsis

ABSTRACTSCF-type E3 ubiquitin ligases use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains. A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of four plant FBX proteins has atypical domain structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or exact biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets of SCFFBS complexes. Furthermore, we found that FBIP1 interacts with NIGT1.1, a GARP-type transcriptional repressor that regulates nitrate and phosphate starvation signaling and responses. Collectively, these interactions between FBS, FBIP, and NIGT1.1 proteins delineate a previously unrecognized SCF-connected transcription regulation module that works in the context of phosphate and nitrate starvation, and possibly other environmental stresses. Importantly, this work also identified two uncharacterized WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

SUSA2 is an F-box protein required for autoimmunity mediated by paired NLRs SOC3-CHS1 and SOC3-TN2

Both higher plants and mammals rely on nucleotide-binding leucine-rich repeat (NLR) immune receptors to detect pathogens and initiate immunity. Upon effector recognition, plant NLRs oligomerize for defense activation, the mechanism of which is poorly understood. We previously showed that disruption of the E3 ligase, Senescence-Associated E3 Ubiquitin Ligase 1 (SAUL1) leads to the activation of the NLR SOC3. Here, we report the identification of suppressor of saul1 2 (susa2) and susa3 from the saul1-1 suppressor screen. Pairwise interaction analysis suggests that both SUSA proteins interact with components of an SCFSUSA2 E3 ligase complex as well as CHS1 or TN2, truncated NLRs that pair with SOC3. susa2-2 only suppresses the autoimmunity mediated by either CHS1 or TN2, suggesting its specific involvement in SOC3-mediated immunity. In summary, our study indicates links between plant NLRs and an SCF complex that may enable ubiquitination and degradation of unknown downstream components to activate defense.