Small Ubiquitin-like Modifier Gene Research Articles

Abstract 348: Elucidation of SUMOylation machinery in human cancer cells and naked mole-rat tissues

Abstract Background: SUMOylation is a post-translational modification where small ubiquitin-like modifier (SUMO) proteins are conjugated to lysine residues of target proteins. SUMO proteins exist in all eukaryotes and participate in a catalytic cycle, which includes maturation, activation, conjugation, ligation, and de-modification. SUMOylation is known to regulate numerous molecular regulatory mechanisms such as: chromatin remodeling, DNA repair, immune responses, cell cycle progression, apoptosis, and the intracellular signal transduction. SUMOylation dysregulation is known to promote several diseases, including cancer cell survival. The unique traits of phenotypic and molecular adaptations found in naked mole rats (NMR) suggest high stability and effective functioning of the molecular machinery that counteract damage accumulation in its genome. NMR lifespan can reach up to 32 years and acquires a very efficient mechanism of resistance to cancer. This study aims to investigate the regulation of SUMO machinery between human cancer cells breast (MCF-7), colorectal (DLD-1), and neuroblastoma (SH-SY5Y)) and NMR brain and intestinal tissues, in order to find novel targets for cancer treatment. Results: The pairwise alignment identified the similarity of both SUMO genes and proteins including SUMO1, SUMO2/3, and SUMO components of Sentrin-specific protease isoforms (SENP 1-7), activation (SAE1 and Uba2), conjugation (Ubc9), and ligation isoforms (PIAS1-4) between NMR and human. In addition, qRT-PCR data revealed low expression of SUMO components at the mRNA level in NMR brain and intestinal tissues compared to MCF-7, DLD-1, and SH-SY5Y cell lines. On the contrary, PIAS4 expression is significantly higher in the NMR tissues compared to cancer cell lines. Protein analysis via Western blotting performed on MCF-7, DLD-1, and SH-SY5Y cell lines revealed similar levels to mRNA compared to NMR tissue. Ongoing studies are focusing on several functional assays to investigate effects of hUBE2I, hSENP1, and Myc/hPIAS4 effects on cytotoxicity and signaling properties of the cancer cells using techniques such as Microculture Tetrazolium (MTT), migration, invasion, and colony formation. The effect on cytotoxicity is also analyzed in relevance to clinical used treatments such as Doxorubicin in MCF-7, 5-Fluorouracil for DLD-1 and Methotrexate in SH-SY5Y. Conclusion: Expression analysis of SUMOylation machinery revealed lower expression of mRNA levels for SUMO components was observed in NMR tissues compared to all human cancer cells. However, PIAS4 expression was significantly higher in the NMR tissues compared to cancer cell lines. This data revealed important differences in NMR SUMOylation machinery (such as UBE2I, SENP1 and PIAS4) which provides a future therapeutic avenue to treat cancer. Citation Format: Mohammed Abdullah Mohammed Salih, Ritchie Williamson, Sriharsha Kantamneni. Elucidation of SUMOylation machinery in human cancer cells and naked mole-rat tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 348.

Sumoylation of Nosema bombycis is essential to its proliferation in silkworm, Bombyx mori

• NbSUMO was successfully cloned and was highly conserved in fungi . • NbSUMO was essential to the proliferation of Nosema Bombycis . • 65 silkworm proteins and 10 Nb proteins may be modified by NbSUMO. Small ubiquitin-like modifier (SUMO) is an important post-translational modification protein, which plays an important role in regulating the function of substrate proteins. The purpose of this study is to explore the function of the SUMO gene in Nosema bombycis (Nb). The SUMO gene of Nb ( NbSUMO ) was successfully cloned and the sequence analysis showed that the amino acid sequence of NbSUMO has a low identity with SUMOs from Homo sapiens and Bombyx mori . Cluster analysis showed that all SUMOs from microsporidium were clustered on the same large branch, and NbSUMO had the highest homology with Nosema pernyi . Real-time fluorescence quantitative PCR (RT-qPCR) analysis showed that the expression of NbSUMO was significantly increased after Nb infection, while the expression of SUMO gene in Bombyx mori ( BmSUMO ) was not, suggesting that Nb relies on its own SUMO modification system to promote proliferation. When the NbSUMO was knocked down by RNA interference (RNAi), the DNA copy of the Nb genome was significantly decreased suggesting NbSUMO was essential to the proliferation of Nb. eGFP-NbSUMO was expressed in BmN cells of Bombyx mori by constructing the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV). Pull-down and mass spectrometry analysis found that 65 silkworm proteins and 10 Nb proteins may bind to NbSUMO, involving in ubiquitin-dependent protein catabolic process, regulation of transcription, apoptotic process, and other biological processes. The study demonstrated that NbSUMO is a typical SUMO family protein. And Nb expresses a large amount of NbSUMO to modify substrate proteins, thus participating in the proliferation of Nb.

Identification and expression analysis of a new small ubiquitin-like modifier from Taenia pisiformis

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

Understanding SUMO-mediated adaptive responses in plants to improve crop productivity.

The response to abiotic and biotic stresses in plants and crops is considered a multifaceted process. Due to their sessile nature, plants have evolved unique mechanisms to ensure that developmental plasticity remains during their life cycle. Among these mechanisms, post-translational modifications (PTMs) are crucial components of adaptive responses in plants and transduce environmental stimuli into cellular signalling through the modulation of proteins. SUMOylation is an emerging PTM that has received recent attention due to its dynamic role in protein modification and has quickly been considered a significant component of adaptive mechanisms in plants during stress with great potential for agricultural improvement programs. In the present review, we outline the concept that small ubiquitin-like modifier (SUMO)-mediated response in plants and crops to abiotic and biotic stresses is a multifaceted process with each component of the SUMO cycle facilitating tolerance to several different environmental stresses. We also highlight the clear increase in SUMO genes in crops when compared with Arabidopsis thaliana. The SUMO system is understudied in crops, given the importance of SUMO for stress responses, and for some SUMO genes, the apparent expansion provides new avenues to discover SUMO-conjugated targets that could regulate beneficial agronomical traits.

Overexpression of GmSUMO2 gene confers increased abscisic acid sensitivity in transgenic soybean hairy roots

Small ubiquitin-like modifier (SUMO) participates in post-translational modification of various target proteins. SUMOylation is an important molecular regulatory mechanism for plants to respond to abiotic stress. In the present study, GmSUMO2 gene was isolated from soybean seedlings for further study because of the highest expression level among these six SUMO genes in soybean. qRT-PCR results showed that GmSUMO2 gene were detected in root, leaf, cotyledon, seed root, flower, pod and seed, with the highest transcription level in cotyledon. Moreover, GmSUMO2 gene was transcriptionally regulated by 200mM NaCl, 42°C, 25μM abscisic acid (ABA) and 20% PEG6000 during the 24h period of treatment. Besides, western blot analysis using AtSUMO1 antibody indicated that the free SUMO levels and SUMOylation dynamics were regulated by ABA stimulus. Functional analysis indicated that overexpression of GmSUMO2 gene in soybean hairy roots accentuated the sensitivity to exogenous ABA. Furthermore, the expression levels of ABI3, ABI5, SnRK1.1 and SnRK1.2 were differentially regulated by GmSUMO2 in transgenic soybean hairy roots. Overall, these results provided a preliminary understanding of molecular characterization, expression and function of GmSUMO2 in soybean.

SUMO-1 Gene Silencing Inhibits Proliferation and Promotes Apoptosis of Human Gastric Cancer SGC-7901 Cells

Background: It has been reported that blocking small ubiquitin-like modifier (SUMO) conjugation by silencing SUMO gene remarkably decreased tumor growth in vivo. However, few studies have examined the relationship between SUMO gene silencing and gastric cancer (GC). The study aims to explore the effects of SUMO-1 gene silencing on GC cell proliferation and apoptosis. Methods: GC cells were cultured and divided into 5 groups: the blank group (without any transfection or treatment), the empty vector group (transfected with empty vector), the shRNA-SUMO-1-1 group (transfected with shRNA-SUMO-1-1 plasmid), the shRNA-SUMO-1-2 group (transfected with shRNA-SUMO-1-2 plasmid), and the shRNA-SUMO-1-3 group (transfected with shRNA-SUMO-1-3 plasmid). Cell Counting Kit-8 (CCK-8) assay was performed to examine cell proliferation. Annexin V/PI staining combined with flow cytometry were used to detect cell apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to measure the mRNA and protein expressions of SUMO-1, P53, Bcl-2 and c-myc, respectively. Results: SUMO-1 mRNA and protein expressions were decreased after transfecting with shRNA-SUMO-1. Compared with the blank group, the shRNA-SUMO-1-1 group presented a remarkable decreased proliferation of SGC-7901 cells. Significant increase in cell apoptosis rate was observed. Bcl-2, c-myc and P53 expressions were declined after transfecting with shRNA-SUMO plasmid. Conclusion: Our study provided evidence that SUMO-1 gene silencing could decrease proliferation and promote apoptosis in GC cells.

Whole-genome duplications followed by tandem duplications drive diversification of the protein modifier SUMO in Angiosperms.

Summary The ubiquitin‐like modifier (UBL) SUMO (Small Ubiquitin‐Like Modifier) regulates protein function. Structural rather than sequence homology typifies UBL families. However, individual UBL types, such as SUMO, show remarkable sequence conservation. Selection pressure also operates at the SUMO gene copy number, as increased SUMO levels activate immunity and alter flowering time in Arabidopsis.We show how, despite this selection pressure, the SUMO family has diversified into eight paralogues in Arabidopsis. Relationships between the paralogues were investigated using genome collinearity and gene tree analysis. We show that palaeopolyploidy followed by tandem duplications allowed expansion and then diversification of the SUMO genes.For example, Arabidopsis SUMO5 evolved from the pan‐eudicot palaeohexaploidy event (gamma), which yielded three SUMO copies. Two gamma copies were preserved as archetype SUMOs, suggesting subfunctionalization, whereas the third copy served as a hotspot for SUMO diversification.The Brassicaceae‐specific alpha duplication then caused the duplication of one archetype gamma copy, which, by subfunctionalization, allowed the retention of both SUMO1 and SUMO2. The other archetype gamma copy was simultaneously pseudogenized (SUMO4/6). A tandem duplication of SUMO2 subsequently yielded SUMO3 in the Brassicaceae crown group. SUMO3 potentially neofunctionalized in Arabidopsis, but it is lost in many Brassicaceae. Our advanced methodology allows the study of the birth and fixation of other paralogues in plants.

Evolution of SUMO Function and Chain Formation in Insects

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.

SUMO (small‐ubiquitin‐like modifier) in Tetrahymena thermophila

Cilia and flagella organelles serve important roles in a wide variety of organisms. Outside of Chlamydomonas reinhardtii flagella, there is limited understanding of ciliary protein composition and regulation. We investigate SUMO (Small ubiquitin‐like modifier) protein modification in the cilia of the single celled protozoan, Tetrahymena thermophila. SUMO is a member of the family of ubiquitin‐like polypeptides that become covalently attached to intracellular target proteins in eukaryotic organisms. Bioinformatics have shown a single SUMO gene in the genome of this organism. Real‐time RT‐PCR of T. thermophila samples has confirmed the expression of this SUMO gene. Additionally, western‐blot analysis of ciliary extracts has shown three SUMOylated proteins of approximately 35kD, 50kD and 135kD. The 135kD SUMOylated protein is preferentially found in ciliary extracts and not in cell body extracts. This implies there are specific SUMOylated target proteins in the cilia. Fractionations of ciliary extracts have been submitted for mass‐spectrometry analysis and reveal proteins not previously found in the T. thermophila ciliary proteome.Supported by NSF and the Nebraska Research Council.

Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.

SUMO (small‐ubiquitin‐like modifier) in subcellular fractions of Chlamydomonas reinhardtii reveal potential function

SUMO (Small ubiquitin‐like modifier), is a member of the family of ubiquitin‐like polypeptides that become covalently attached to intracellular target proteins. SUMOylation can alter the function, location, and/or half‐life of target proteins. Here we describe three SUMO proteins and three SUMO‐like proteins in Chlamydomonas reinhardtii. The three SUMO genes show differential RNA expression depending on environmental conditions. Western analysis also shows a shift in proteins recognized by the Chlamydomonas reinhardtii SUMO antibodies in response to changes in environmental conditions. The three SUMO‐like proteins are novel fusion proteins with C‐terminal SUMO. Chlamydomonas reinhardtii subcellular fractionation reveals SUMO proteins are conjugated to proteins involved in various cellular processes. Proposed SUMOylated target proteins can be resolved by one‐dimensional and two‐dimensional western analysis.Supported by NSF and the Nebraska Research Council.

Molecular features of human ubiquitin-like SUMO genes and their encoded proteins

The SUMO (small ubiquitin-like modifier) protein and ubiquitin have similar 3-D structure. Sumolyzation and ubiquitination exhibit similar biological processes for post-translational modification. However, unlike ubiquitination, which targets proteins for degradation, sumolyzation participates in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and protein stability. The human genome contains three SUMO-1/2/3 functional genes, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes, but no SUMO-3 pseudogenes. The protein-coding sequence of the SUMO-1 gene is interrupted by four introns, while those of SUMO-2 and SUMO-3 genes are interrupted by three introns. Human SUMO-1 protein exhibits 44% sequence identity with SUMO-2 and SUMO-3 proteins, while SUMO-2 and SUMO-3 proteins share 86% sequence identity. Phylogenetic analyses indicate that the SUMO-3 gene was derived from the SUMO-2 gene. SUMO-1 mRNA appears to be most abundant in human epithelial HeLa, kidney 293 and neuronal NT2 cells, while the SUMO-3 mRNA seems to be much less abundant than SUMO-2 mRNA, especially in HeLa and 293 cells. Many cellular proteins of high molecular weights were covalently modified by SUMO-1/2/3 proteins. However, some free form of SUMO-2/3 proteins was also detected. Most SUMO-1/2/3 proteins were shown to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively.