Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas

*Xianquan Zhan and Miaolong Lu*

#### **Abstract**

Ubiquitination is an important post-translational modification. Abnormal ubiquitination is extensively associated with cancers. Lung squamous cell carcinoma (LUSC) is the most common pathological type of lung cancer, with unclear molecular mechanism and the poor overall prognosis of LUSC patient. To uncover the existence and potential roles of ubiquitination in LUSC, label-free quantitative ubiquitomics was performed in human LUSC vs. control tissues. In total, 627 ubiquitinated proteins (UPs) with 1209 ubiquitination sites were identified, including 1133 (93.7%) sites with quantitative information and 76 (6.3%) sites with qualitative information. KEGG pathway enrichment analysis found that UPs were significantly enriched in ubiquitin-mediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). Further analysis of 400 differentially ubiquitinated proteins (DUPs) revealed that 11 subunits of the proteasome complex were differentially ubiquitinated. These findings clearly demonstrated that ubiquitination was widely present in the ubiquitin-proteasome pathway in LUSCs. At the same time, abnormal ubiquitination might affect the function of the proteasome to promote tumorigenesis and development. This book chapter discussed the status of protein ubiquitination in the ubiquitin-proteasome system (UPS) in human LUSC tissues, which offered the scientific data to elucidate the specific molecular mechanisms of abnormal ubiquitination during canceration and the development of anti-tumor drugs targeting UPS.

**Keywords:** lung squamous cell carcinoma, ubiquitination, ubiquitinated protein (UP), differentially ubiquitinated protein (DUP), ubiquitin-proteasome system (UPS)

#### **1. Introduction**

Ubiquitination is one of the important protein post-translational modifications (PTMs) in human body, in which ubiquitin, a 76-amino-acid protein with a molecular weight of 8.5 KDa, is covalently attached its C-terminus to the ε-amino group of the substrate protein lysine residue through a multi-step enzymatic reaction cascade catalyzed by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) [1]. As substrate proteins commonly contain multiple lysine residues, there are a variety of ubiquitination forms such as monoubiquitination (only one ubiquitin attached to a protein), multiubiquitination

(several lysine residues of substrate proteins were tagged with single ubiquitin), and polyubiquitination (a polyubiquitin chain is derived from subsequent ubiquitin covalently attached to lysine residues or N-terminus of the former ubiquitin) [2]. It is worth noting that the ubiquitin itself also has seven lysine residues to greatly complicate the topology of the polyubiquitin chain. Different ubiquitination forms perform different functions, such as monoubiquitination or multiubiquitination has been shown to be required for the entry of certain cargo proteins into vesicles at different stages of the secretory/endocytic pathway, while lysine-48 ubiquitin chain is mainly related to proteasome [3]. Like other PTMs, ubiquitination is a reversible reaction, and there are over 100 deubiquitination enzymes that regulate this process [4]. Ubiquitination coordinates with deubiquitination to regulate a broad host of cellular processes, including DNA repair, cell differentiation, signal transduction, enzymatic activity regulation, assembly of multiprotein complexes, protein trafficking, and autophagy [5]. Therefore, abnormal ubiquitination is associated with many diseases, including cancer, neurodegenerative disease, infection, and immune disorders [6]. Considering the importance of ubiquitination in tumorigenesis, different components of ubiquitin-proteasome system could be regarded as targets for discovery of anti-tumor drugs. With the application of first and second therapeutic proteasome inhibitors, such as Bortezomib (FDA has approved it for multiple myeloma and mantle cell lymphoma) [7] and Carfilzomib (FDA has approved it for relapsed and refractory multiple myeloma) [8], more and more anti-tumor drugs targeting UPS have been developed and approved by FDA, such as thalidomide, lenalidomide, and pomalidomide for treatment of multiple myeloma [9, 10].

Lung squamous cell carcinoma (LUSC) is a common type of lung cancer without a clear molecular mechanism. Currently, surgery, radiation, and chemotherapy have made significant advances in lung cancer treatment, especially targeted drug therapy; for example, epidermal growth factor receptor (EGFR) mutation or EML4-ALK fusion-based targeted therapies have improved the survival time of patients with lung adenocarcinoma (LUAD). However, targeted therapy and earlystage diagnosis are still a big clinical challenge in LUSC patients [11]. Although FGFR1 amplification and DDR2 mutation have been nominated as "druggable" targets in LUSC patients, the clinical efficacies of the corresponding drugs are still under clinical trials [12, 13]. Considering that abnormal ubiquitination will lead to the occurrence of a variety of tumors and the widespread clinical applications of anti-tumor drugs for the ubiquitin-proteasome pathway in recent years, the study of quantitative ubiquitinomics in LUSC tissues may provide the direction for the development of biomarkers and new targeted drugs.

High-resolution liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS) has been used as a power tool for large-scale identification of various PTMs such as ubiquitination, phosphorylation, acetylation, and N-glycosylation [14]. The challenge of the use of LC-MS/MS to identify endogenous ubiquitination sites on a large scale is the fact that ubiquitination is a low abundance event *in vivo* and the size of the modification itself. The common strategy to identify low-abundance ubiquitinations in a proteome is that the extracted protein sample is firstly digested with trypsin to form tryptic peptide mixture, then the commercially specific anti-K-ε-GG antibodies are used to preferentially enrich ubiquitinated peptides from tryptic peptide mixture before MS/MS analysis in recent years [15]. Anti-ubiquitin antibody (specific anti-K-ε-GG group)-based label-free quantification coupled with LC-MS/MS has been used as an effective method to detect, identify, and quantify ubiquitinated proteins and ubiquitination sites, and more than 10,000 ubiquitination sites have been identified and quantified [16]. For lung cancer, ubiquitinomics is mainly carried out in lung cancer cells [17, 18], while the ubiquitinomics of fresh LUSC tissues is only reported recently in our research

**97**

**2.3 LC-MS/MS**

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

group with label-free quantitative proteomics method and bioinformatics analysis to reveal the functions of ubiquitinome in predictive, preventive, and personalized

This book chapter mainly reviewed ubiquitinated proteins (UPs) and differentially ubiquitinated proteins (DUPs) in ubiquitin-proteasome-system (UPS) in LUSC, and emphasized the potential regulatory role of ubiquitination in UPS, which offers scientific data for further research on the regulatory mechanism of ubiquitination on UPS, the molecular mechanism of UPS abnormality in tumor

Human LUSC tissues (n = 5) and tumor-adjacent control lung tissues (n = 5) were surgically removed from patients, immediately stored in liquid nitrogen (−196°C), and then stored in freezer (−80°C). Clinical characteristics of each sample were described previously [19]. LUSC tissues (750 mg, equally mixed 5 tumor tissues) and control tissues (750 mg, equally mixed 5 control tissues) were washed 5 times with 3 mL 0.9% NaCl to clean blood on the surface of the tissues. The washed tissues (LUSC; or controls) were homogenized with urea lysis buffer [2 M thiourea, 7 M urea, 1 mM protein inhibitor PMSF, and 100 mM dithiothreitol(DTT)], sonicated, and centrifuged (15,000 g, 20 min, and 4°C). The supernatant was the extracted protein sample. The protein content was tested with Brandford method. The detailed

An amount of DTT (final concentration = 10 mM) was added to each extracted

The prepared ubiquitinated peptide sample was analyzed with LC-MS/MS in the Easy nLC and Q Exactive mass spectrometer (Thermo Scientific). The MS/MS data for each sample were used to search protein database using MaxQuant 1.5.3.17 software to identify ubiquitinated proteins and ubiquitination sites and quantify the abundance of ubiquitination. The detailed procedure was described previously [19].

protein sample, which was mixed (600 rpm, 1.5 h, and room temperature). An amount of iodoacetamide (final concentration = 50 mM) was added to the DTT-treated protein sample, which was incubated (dark, 30 min). The uranyl acetate (UA) was diluted to 2 M with 50 mM Tris HCl buffer (pH 8.0) and added to each protein sample. An amount of trypsin was added to each protein sample (trypsin:protein = 1:50 at wt:wt), and then incubated (37°C, 15–18 h). A volume of 10% trifluoroacetic acid (TFA; final concentration = 0.1%) was added, and pH was adjusted to ≤ 3 to stop digestion. Each tryptic peptide sample was purified with C18 cartridges and then lyophilized. The lyophilized tryptic peptides were resolved with 1.4 mL immunoaffinity purification (IAP) buffer that contained 50 mM NaCl, 10 mM Na2HPO4, 50 mM MOPS/NaOH, and pH 7.2. The anti-K-ε-GG antibodies against ubiquitin remnant motif (K-ε-GG) (Cell Signal Technology) were used to enrich the ubiquitinated peptides, followed by purification with C18 STAGE Tips. The purified ubiquitinated peptide sample was used for MS/MS analysis. The

development, and the development of anti-tumor drugs targeting UPS.

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

medicine (PPPM) of LUSC [19].

**2. Materials and methods**

**2.1 Lung cancer tissues and protein extraction**

procedure of protein extraction was described previously [19].

**2.2 Protein digestion and enrichment of ubiquitinated peptides**

detailed experimental procedure was described previously [19].

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*

group with label-free quantitative proteomics method and bioinformatics analysis to reveal the functions of ubiquitinome in predictive, preventive, and personalized medicine (PPPM) of LUSC [19].

This book chapter mainly reviewed ubiquitinated proteins (UPs) and differentially ubiquitinated proteins (DUPs) in ubiquitin-proteasome-system (UPS) in LUSC, and emphasized the potential regulatory role of ubiquitination in UPS, which offers scientific data for further research on the regulatory mechanism of ubiquitination on UPS, the molecular mechanism of UPS abnormality in tumor development, and the development of anti-tumor drugs targeting UPS.

#### **2. Materials and methods**

*Ubiquitin - Proteasome Pathway*

(several lysine residues of substrate proteins were tagged with single ubiquitin), and polyubiquitination (a polyubiquitin chain is derived from subsequent ubiquitin covalently attached to lysine residues or N-terminus of the former ubiquitin) [2]. It is worth noting that the ubiquitin itself also has seven lysine residues to greatly complicate the topology of the polyubiquitin chain. Different ubiquitination forms perform different functions, such as monoubiquitination or multiubiquitination has been shown to be required for the entry of certain cargo proteins into vesicles at different stages of the secretory/endocytic pathway, while lysine-48 ubiquitin chain is mainly related to proteasome [3]. Like other PTMs, ubiquitination is a reversible reaction, and there are over 100 deubiquitination enzymes that regulate this process [4]. Ubiquitination coordinates with deubiquitination to regulate a broad host of cellular processes, including DNA repair, cell differentiation, signal transduction, enzymatic activity regulation, assembly of multiprotein complexes, protein trafficking, and autophagy [5]. Therefore, abnormal ubiquitination is associated with many diseases, including cancer, neurodegenerative disease, infection, and immune disorders [6]. Considering the importance of ubiquitination in tumorigenesis, different components of ubiquitin-proteasome system could be regarded as targets for discovery of anti-tumor drugs. With the application of first and second therapeutic proteasome inhibitors, such as Bortezomib (FDA has approved it for multiple myeloma and mantle cell lymphoma) [7] and Carfilzomib (FDA has approved it for relapsed and refractory multiple myeloma) [8], more and more anti-tumor drugs targeting UPS have been developed and approved by FDA, such as thalidomide, lenalidomide, and pomalidomide for treatment of multiple myeloma [9, 10].

Lung squamous cell carcinoma (LUSC) is a common type of lung cancer without

a clear molecular mechanism. Currently, surgery, radiation, and chemotherapy have made significant advances in lung cancer treatment, especially targeted drug therapy; for example, epidermal growth factor receptor (EGFR) mutation or EML4-ALK fusion-based targeted therapies have improved the survival time of patients with lung adenocarcinoma (LUAD). However, targeted therapy and earlystage diagnosis are still a big clinical challenge in LUSC patients [11]. Although FGFR1 amplification and DDR2 mutation have been nominated as "druggable" targets in LUSC patients, the clinical efficacies of the corresponding drugs are still under clinical trials [12, 13]. Considering that abnormal ubiquitination will lead to the occurrence of a variety of tumors and the widespread clinical applications of anti-tumor drugs for the ubiquitin-proteasome pathway in recent years, the study of quantitative ubiquitinomics in LUSC tissues may provide the direction for the

High-resolution liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS) has been used as a power tool for large-scale identification of various PTMs such as ubiquitination, phosphorylation, acetylation, and N-glycosylation [14]. The challenge of the use of LC-MS/MS to identify endogenous ubiquitination sites on a large scale is the fact that ubiquitination is a low abundance event *in vivo* and the size of the modification itself. The common strategy to identify low-abundance ubiquitinations in a proteome is that the extracted protein sample is firstly digested with trypsin to form tryptic peptide mixture, then the commercially specific anti-K-ε-GG antibodies are used to preferentially enrich ubiquitinated peptides from tryptic peptide mixture before MS/MS analysis in recent years [15]. Anti-ubiquitin antibody (specific anti-K-ε-GG group)-based label-free quantification coupled with LC-MS/MS has been used as an effective method to detect, identify, and quantify ubiquitinated proteins and ubiquitination sites, and more than 10,000 ubiquitination sites have been identified and quantified [16]. For lung cancer, ubiquitinomics is mainly carried out in lung cancer cells [17, 18], while the ubiquitinomics of fresh LUSC tissues is only reported recently in our research

development of biomarkers and new targeted drugs.

**96**

#### **2.1 Lung cancer tissues and protein extraction**

Human LUSC tissues (n = 5) and tumor-adjacent control lung tissues (n = 5) were surgically removed from patients, immediately stored in liquid nitrogen (−196°C), and then stored in freezer (−80°C). Clinical characteristics of each sample were described previously [19]. LUSC tissues (750 mg, equally mixed 5 tumor tissues) and control tissues (750 mg, equally mixed 5 control tissues) were washed 5 times with 3 mL 0.9% NaCl to clean blood on the surface of the tissues. The washed tissues (LUSC; or controls) were homogenized with urea lysis buffer [2 M thiourea, 7 M urea, 1 mM protein inhibitor PMSF, and 100 mM dithiothreitol(DTT)], sonicated, and centrifuged (15,000 g, 20 min, and 4°C). The supernatant was the extracted protein sample. The protein content was tested with Brandford method. The detailed procedure of protein extraction was described previously [19].

#### **2.2 Protein digestion and enrichment of ubiquitinated peptides**

An amount of DTT (final concentration = 10 mM) was added to each extracted protein sample, which was mixed (600 rpm, 1.5 h, and room temperature). An amount of iodoacetamide (final concentration = 50 mM) was added to the DTT-treated protein sample, which was incubated (dark, 30 min). The uranyl acetate (UA) was diluted to 2 M with 50 mM Tris HCl buffer (pH 8.0) and added to each protein sample. An amount of trypsin was added to each protein sample (trypsin:protein = 1:50 at wt:wt), and then incubated (37°C, 15–18 h). A volume of 10% trifluoroacetic acid (TFA; final concentration = 0.1%) was added, and pH was adjusted to ≤ 3 to stop digestion. Each tryptic peptide sample was purified with C18 cartridges and then lyophilized. The lyophilized tryptic peptides were resolved with 1.4 mL immunoaffinity purification (IAP) buffer that contained 50 mM NaCl, 10 mM Na2HPO4, 50 mM MOPS/NaOH, and pH 7.2. The anti-K-ε-GG antibodies against ubiquitin remnant motif (K-ε-GG) (Cell Signal Technology) were used to enrich the ubiquitinated peptides, followed by purification with C18 STAGE Tips. The purified ubiquitinated peptide sample was used for MS/MS analysis. The detailed experimental procedure was described previously [19].

#### **2.3 LC-MS/MS**

The prepared ubiquitinated peptide sample was analyzed with LC-MS/MS in the Easy nLC and Q Exactive mass spectrometer (Thermo Scientific). The MS/MS data for each sample were used to search protein database using MaxQuant 1.5.3.17 software to identify ubiquitinated proteins and ubiquitination sites and quantify the abundance of ubiquitination. The detailed procedure was described previously [19].

#### **2.4 Bioinformatics**

For UPs and DUPs, DAVID software (version 6.8, https://david.ncifcrf.gov/) was used to carry out the gene ontology (GO) enrichment analysis, including cellular components (CCs), molecular functions (MFs), and biological processes (BPs), and group those proteins into different functional clusters [20]. The statistically significant pathways were mined with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The KEGG online service tool KOBAS (http://kobas.cbi. pku.cn) was used to annotate the KEGG database description of each protein [21].

#### **3. Results and discussion**

#### **3.1 Proteomics analysis of lysine-ubiquitinated profile in LUSC**

To identify protein lysine-ubiquitinated sites and quantify the level of ubiquitination in human LUSC tissues, proteins were extracted and digested into peptides with trypsin. Lysine-ubiquitinated peptides were immunoaffinityenriched with commercially specific anti-K-ε-GG antibodies and analyzed with high-resolution LC-MS/MS. In total, 1209 lysine-ubiquitinated sites in 627 unique proteins were identified. These proteins containing ubiquitinated lysine residues were defined as UPs. **Figure 1** showed two representative MS/MS spectra of the ubiquitinated peptides 425ETNLDSLPLVDTHSK\*R440 from vimentin (P08670; K\* = ubiquitinated lysine residue) (**Figure 1A**), and 633RPVK\*DGGGTNSITVR647 from multidrug resistance-associated protein 1 (P33527; K\* = ubiquitinated lysine residue) (**Figure 1B**). All other ubiquitinated sites and ubiquitinated proteins were identified with the same MS/MS method. The differentially ubiquitinated peptides were determined with amino acid sequences, ratio(tumor/control) > 2.0 or < 0.5, and p-value < 0.05. Proteins containing this type of ubiquitinated peptides were defined as DUPs. Totally, 400 DUPs with 654 ubiquitinated sites were identified in LUSC tissues vs. tumor-adjacent control lung tissues [19].

#### **3.2 UPs and DUPs were significantly enriched in UPS-related biological processes and molecular functions in LUSC**

GO functional enrichment analyses of 627 UPs and 400 DUPs were carried out according to BPs, MFs, and CCs. GO enrichment result-based cluster analysis grouped those UPs into seven clusters (**Table 1**), and DUPs into 10 clusters (**Table 2**).

Among GO enrichment results of 627 UPs, many biological processes, molecular functions, and cellular components related to UPS were significantly enriched, including negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle, and positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition in cluster 2, and proteasome-activating ATPase activity, positive regulation of proteasomal protein catabolic process, cytosolic proteasome complex, nuclear proteasome complex, and proteasome regulatory base complex in cluster 3 (**Table 1**). Interestingly, DUPs were also significantly enriched in the similar biological processes, molecular functions, and cellular components, including proteasome accessory complex, negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle, positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition, and protein polyubiquitination in cluster 2, and proteasome-activating ATPase activity, positive regulation of proteasomal protein catabolic process, cytosolic proteasome complex, nuclear proteasome complex, and proteasome regulatory

**99**

development of LUSC.

*publisher open access article, copyright 2020.*

**Figure 1.**

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

base complex in cluster 3 (**Table 2**). These findings clearly demonstrated that many ubiquitinated proteins were involved in UPS system, and differential ubiquitination occurred in UPS system in LUSC, implying that ubiquitination participated in the regulation of UPS, and abnormal ubiquitination might play an important role in the

*Representative MS/MS spectra of ubiquitinated peptides 425ETNLDSLPLVDTHSK\*R440 from vimentin (P08670) (A) and 633RPVK\*DGGGTNSITVR647 from multidrug resistance-associated protein 1 (MRP1) (P33527) (B). K\* = ubiquitinated lysine residue. Reproduced from Lu et al. [19], with permission from Springer* 

KEGG pathway network analysis of 627 UPs revealed 47 statistically significant ubiquitination-mediated signaling pathway alterations (P < 0.05 and FDR < 0.05) (**Figure 2**), among which were included two UPS-related pathways—ubiquitinmediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). Ubiquitin-mediated proteolysis pathway showed the detailed process of protein ubiquitination, which involved multiple types of E1s, E2s, and E3s. This study found that one E1 (UBE1), two E2s (UBE2N, and UBE2O), and six E3s (ITCH, HUWE1, UBE4B, PML, CUL4A, and CUL5) were ubiquitinated in LUSC (**Figure 3**). These six E3s belonged to different subfamilies, in which ITCH and HUWE1 belonged to HECT type E3, UBE4B belonged to U-box type E3, PML belonged to single RINGfinger type E3, and both CUL4A and CUL5 belonged to multi subunit RING-finger type E3. E1s, E2s, and E3s are the important enzymes to catalyze the occurrence of ubiquitination in a protein. The ubiquitination of these enzymes definitely affects the quitination process of a protein. Currently, studies on these enzymes have focused on their roles in the ubiquitination process, and the effects of PTMs on these enzymes are poorly understood. There are relatively few studies on the ubiquitination of these nine enzymes. For example, ubiquitinated PML (P29590, identified in this study) was mediated by multiple E3s, leading to subsequent proteasomal degradation [22, 23]. Self-ubiquitination of ITCH (Q96J02, identified in this study)

**3.3 UPs involved in UPS-related molecular network alternations in LUSC**

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*

**Figure 1.**

*Ubiquitin - Proteasome Pathway*

**3. Results and discussion**

For UPs and DUPs, DAVID software (version 6.8, https://david.ncifcrf.gov/) was used to carry out the gene ontology (GO) enrichment analysis, including cellular components (CCs), molecular functions (MFs), and biological processes (BPs), and group those proteins into different functional clusters [20]. The statistically significant pathways were mined with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The KEGG online service tool KOBAS (http://kobas.cbi. pku.cn) was used to annotate the KEGG database description of each protein [21].

**3.1 Proteomics analysis of lysine-ubiquitinated profile in LUSC**

LUSC tissues vs. tumor-adjacent control lung tissues [19].

**processes and molecular functions in LUSC**

**3.2 UPs and DUPs were significantly enriched in UPS-related biological** 

GO functional enrichment analyses of 627 UPs and 400 DUPs were carried out according to BPs, MFs, and CCs. GO enrichment result-based cluster analysis grouped those UPs into seven clusters (**Table 1**), and DUPs into 10 clusters (**Table 2**). Among GO enrichment results of 627 UPs, many biological processes, molecular

functions, and cellular components related to UPS were significantly enriched, including negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle, and positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition in cluster 2, and proteasome-activating ATPase activity, positive regulation of proteasomal protein catabolic process, cytosolic proteasome complex, nuclear proteasome complex, and proteasome regulatory base complex in cluster 3 (**Table 1**). Interestingly, DUPs were also significantly enriched in the similar biological processes, molecular functions, and cellular components, including proteasome accessory complex, negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle, positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition, and protein polyubiquitination in cluster 2, and proteasome-activating ATPase activity, positive regulation of proteasomal protein catabolic process, cytosolic proteasome complex, nuclear proteasome complex, and proteasome regulatory

To identify protein lysine-ubiquitinated sites and quantify the level of ubiquitination in human LUSC tissues, proteins were extracted and digested into peptides with trypsin. Lysine-ubiquitinated peptides were immunoaffinityenriched with commercially specific anti-K-ε-GG antibodies and analyzed with high-resolution LC-MS/MS. In total, 1209 lysine-ubiquitinated sites in 627 unique proteins were identified. These proteins containing ubiquitinated lysine residues were defined as UPs. **Figure 1** showed two representative MS/MS spectra of the ubiquitinated peptides 425ETNLDSLPLVDTHSK\*R440 from vimentin (P08670; K\* = ubiquitinated lysine residue) (**Figure 1A**), and 633RPVK\*DGGGTNSITVR647 from multidrug resistance-associated protein 1 (P33527; K\* = ubiquitinated lysine residue) (**Figure 1B**). All other ubiquitinated sites and ubiquitinated proteins were identified with the same MS/MS method. The differentially ubiquitinated peptides were determined with amino acid sequences, ratio(tumor/control) > 2.0 or < 0.5, and p-value < 0.05. Proteins containing this type of ubiquitinated peptides were defined as DUPs. Totally, 400 DUPs with 654 ubiquitinated sites were identified in

**2.4 Bioinformatics**

**98**

*Representative MS/MS spectra of ubiquitinated peptides 425ETNLDSLPLVDTHSK\*R440 from vimentin (P08670) (A) and 633RPVK\*DGGGTNSITVR647 from multidrug resistance-associated protein 1 (MRP1) (P33527) (B). K\* = ubiquitinated lysine residue. Reproduced from Lu et al. [19], with permission from Springer publisher open access article, copyright 2020.*

base complex in cluster 3 (**Table 2**). These findings clearly demonstrated that many ubiquitinated proteins were involved in UPS system, and differential ubiquitination occurred in UPS system in LUSC, implying that ubiquitination participated in the regulation of UPS, and abnormal ubiquitination might play an important role in the development of LUSC.

#### **3.3 UPs involved in UPS-related molecular network alternations in LUSC**

KEGG pathway network analysis of 627 UPs revealed 47 statistically significant ubiquitination-mediated signaling pathway alterations (P < 0.05 and FDR < 0.05) (**Figure 2**), among which were included two UPS-related pathways—ubiquitinmediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050).

Ubiquitin-mediated proteolysis pathway showed the detailed process of protein ubiquitination, which involved multiple types of E1s, E2s, and E3s. This study found that one E1 (UBE1), two E2s (UBE2N, and UBE2O), and six E3s (ITCH, HUWE1, UBE4B, PML, CUL4A, and CUL5) were ubiquitinated in LUSC (**Figure 3**). These six E3s belonged to different subfamilies, in which ITCH and HUWE1 belonged to HECT type E3, UBE4B belonged to U-box type E3, PML belonged to single RINGfinger type E3, and both CUL4A and CUL5 belonged to multi subunit RING-finger type E3. E1s, E2s, and E3s are the important enzymes to catalyze the occurrence of ubiquitination in a protein. The ubiquitination of these enzymes definitely affects the quitination process of a protein. Currently, studies on these enzymes have focused on their roles in the ubiquitination process, and the effects of PTMs on these enzymes are poorly understood. There are relatively few studies on the ubiquitination of these nine enzymes. For example, ubiquitinated PML (P29590, identified in this study) was mediated by multiple E3s, leading to subsequent proteasomal degradation [22, 23]. Self-ubiquitination of ITCH (Q96J02, identified in this study)


#### **Table 1.**

*The functional categories of 627 ups, identified with GO enrichment analysis.*

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*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

**Category GO term p-value**

GOTERM\_MF\_DIRECT Cadherin binding involved in cell-cell adhesion 3.11E-18 GOTERM\_CC\_DIRECT Cell-cell adherens junction 2.24E-17 GOTERM\_BP\_DIRECT Cell-cell adhesion 1.23E-13

GOTERM\_CC\_DIRECT Proteasome accessory complex 4.60E-12

GOTERM\_BP\_DIRECT Regulation of cellular amino acid metabolic process 3.49E-09 GOTERM\_BP\_DIRECT NIK/NF-kappaB signaling 5.35E-09

GOTERM\_BP\_DIRECT Stimulatory C-type lectin receptor signaling pathway 1.85E-08

GOTERM\_BP\_DIRECT Tumor necrosis factor-mediated signaling pathway 8.37E-08 GOTERM\_BP\_DIRECT Positive regulation of canonical Wnt signaling pathway 1.04E-07 GOTERM\_BP\_DIRECT T cell receptor signaling pathway 2.37E-07 GOTERM\_BP\_DIRECT Fc-epsilon receptor signaling pathway 4.90E-07 GOTERM\_BP\_DIRECT Protein polyubiquitination 7.65E-07 GOTERM\_BP\_DIRECT Negative regulation of canonical Wnt signaling pathway 4.36E-06

GOTERM\_CC\_DIRECT Proteasome regulatory particle, base subcomplex 4.88E-10 GOTERM\_CC\_DIRECT Nuclear proteasome complex 1.00E-09 GOTERM\_CC\_DIRECT Cytosolic proteasome complex 7.28E-09 GOTERM\_MF\_DIRECT Proteasome-activating ATPase activity 1.54E-08

GOTERM\_MF\_DIRECT TBP-class protein binding 2.10E-06

GOTERM\_BP\_DIRECT Protein catabolic process 1.41E-04

cell-Purkinje myocyte communication

GOTERM\_CC\_DIRECT Haptoglobin-hemoglobin complex 2.43E-05 GOTERM\_CC\_DIRECT Endocytic vesicle lumen 1.71E-04 GOTERM\_MF\_DIRECT Haptoglobin binding 1.10E-03 GOTERM\_BP\_DIRECT Positive regulation of cell death 1.90E-02

GOTERM\_BP\_DIRECT Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly

GOTERM\_BP\_DIRECT Positive regulation of proteasomal protein catabolic process

GOTERM\_BP\_DIRECT Regulation of ventricular cardiac muscle cell action potential

GOTERM\_MF\_DIRECT Cell adhesive protein binding involved in bundle of His

peptide antigen via MHC class I, TAP-dependent

involved in regulation of mitotic cell cycle transition

1.59E-11

1.27E-08

2.83E-08

4.43E-08

3.41E-07

1.05E-05

1.11E-03

5.30E-03

GOTERM\_BP\_DIRECT Antigen processing and presentation of exogenous

GOTERM\_BP\_DIRECT Negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle

GOTERM\_BP\_DIRECT Positive regulation of ubiquitin-protein ligase activity

GOTERM\_BP\_DIRECT Anaphase-promoting complex-dependent catabolic process

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

**Annotation cluster 1**

**Annotation cluster 2**

**Annotation cluster 3**

**Annotation cluster 4**

**Annotation cluster 5**

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*


*Ubiquitin - Proteasome Pathway*

**Annotation cluster 1**

**Annotation cluster 2**

**Annotation cluster 3**

**Annotation cluster 4**

**Annotation cluster 5**

**Annotation cluster 6**

**Annotation cluster 7**

**Category GO term p -value**

GOTERM\_MF\_DIRECT Cadherin binding involved in cell-cell adhesion 1.43E-29 GOTERM\_CC\_DIRECT Cell-cell adherens junction 3.72E-29 GOTERM\_BP\_DIRECT Cell-cell adhesion 1.13E-23

GOTERM\_BP\_DIRECT Wnt signaling pathway, planar cell polarity pathway 5.78E-11 GOTERM\_BP\_DIRECT Regulation of cellular amino acid metabolic process 4.14E-09 GOTERM\_BP\_DIRECT Stimulatory C-type lectin receptor signaling pathway 4.43E-09 GOTERM\_BP\_DIRECT NIK/NF-kappa B signaling 1.37E-08 GOTERM\_BP\_DIRECT Tumor necrosis factor-mediated signaling pathway 2.96E-08

involved in regulation of mitotic cell cycle transition

GOTERM\_BP\_DIRECT T cell receptor signaling pathway 9.74E-07 GOTERM\_BP\_DIRECT Positive regulation of canonical Wnt signaling pathway 1.13E-06 GOTERM\_BP\_DIRECT Negative regulation of canonical Wnt signaling pathway 1.57E-05

GOTERM\_CC\_DIRECT Proteasome regulatory particle, base subcomplex 1.29E-08 GOTERM\_CC\_DIRECT Nuclear proteasome complex 1.70E-08 GOTERM\_CC\_DIRECT Cytosolic proteasome complex 1.21E-07 GOTERM\_MF\_DIRECT Proteasome-activating ATPase activity 1.70E-07

transcriptional preinitiation complex assembly

GOTERM\_MF\_DIRECT TBP-class protein binding 3.27E-05

GOTERM\_BP\_DIRECT Viral transcription 7.74E-08

GOTERM\_BP\_DIRECT Translational initiation 6.60E-06 GOTERM\_CC\_DIRECT Ribosome 3.32E-05 GOTERM\_MF\_DIRECT Structural constituent of ribosome 1.68E-03

GOTERM\_CC\_DIRECT Haptoglobin-hemoglobin complex 9.95E-05

GOTERM\_BP\_DIRECT Nucleotide-excision repair, DNA damage recognition 6.40E-04

GOTERM\_CC\_DIRECT Hemoglobin complex 4.59E-03

3.70E-08

9.13E-08

1.52E-07

3.45E-06

9.97E-05

3.56E-08

1.92E-07

GOTERM\_BP\_DIRECT Negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle

GOTERM\_BP\_DIRECT Positive regulation of ubiquitin-protein ligase activity

GOTERM\_BP\_DIRECT Anaphase-promoting complex-dependent catabolic

process

GOTERM\_BP\_DIRECT Positive regulation of RNA polymerase II

process

GOTERM\_BP\_DIRECT Positive regulation of proteasomal protein catabolic

GOTERM\_BP\_DIRECT SRP-dependent cotranslational protein targeting to membrane

GOTERM\_BP\_DIRECT Nuclear-transcribed mRNA catabolic process, nonsense-mediated decay

*The functional categories of 627 ups, identified with GO enrichment analysis.*

**100**

**Table 1.**


#### **Table 2.**

*The functional categories of 400 DUPs, identified with GO enrichment analysis. Modified from Lu et al. [19], with permission from Springer publisher open access article, copyright 2020.*

through lysine-63 linkages showed an auto-regulatory mechanism controlling ITCH cytoplasmic-nuclear shuffling [24]. Therefore, the effects of the currently known ubiquitination on these enzymes are only the tip of the iceberg. However, one should also realize that this study found ubiquitination of cullin proteins such as Cul4A and Cul5, while cullin proteins can also be modified by NEDD8 to form NEDDylation. It is well known that the use of the K-ε-GG antibody cannot discriminate between proteins modified with ubiquitin and the related proteins NEDD8 and ISG15. Therefore, for deep investigation of this identified ubiquitination of E3s Cul4A and Cul5 in LUSC in the future, additional experiments are needed to discriminate E3s Cul4A and Cul5 that were modified by ubiquitin, NEDD8, or ISG15 [22].

Proteasome was a pivotal component for ubiquitin-mediated proteolysis. The 26S proteasome was a complex including two 19S regulatory particles (PA700) and one 20S core particle. The 20S degradation complex contained two α rings (7 subunits, α1-α7) and two β rings (7 subunits, β1-β7). These α rings and β rings together formed a hollow ground circle. The tube-like structure was highly conserved

**103**

**Figure 2.**

*the pathway.*

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

from archaea to mammals [25]. Among them, the α ring was located in the outer layer of the cylinder-like structure, which mainly acted on the recognition of the substrate; the β ring was located in the inner layer of the cylinder-like structure, and was mainly responsible for catalyzing the degradation of the substrate [26, 27]. Three subunits that played a catalytic role were located on the inner surface of the β-ring molecule, exhibiting cysteine protease-like activity, trypsin-like activity, and chymotrypsin-like activity [26, 27]. The 19S regulatory complex contained 19 different subunits, which were divided into two parts: "base" and "lid" [28]. Among them, the base part formed the proximal part of the 19S regulatory complex, which was connected to the alpha ring of the 20S degradation complex, and the lip part formed the distal end. The base section contained 6 ATPase-dependent subunits (Rpt1-Rpt6) and 2 ATPase-independent subunits (Rpn1 and Rpn2) [28]. Usually, Rpn10 and Rpn13 were also classified as the base [26]. The lid part consisted of Rpn3, Rpn5-Rpn9, and Rpn11-Rpn12 subunits [28]. The 19S regulatory complex recognized ubiquitin-labeled target proteins (Rpn10 and Rpn13) and before the target protein entered the 20S degradation complex, deubiquitinated the target protein (Rpn11) and opened the folded structure of the target protein [28]. This study discovered five UPs (Rpn3, Rpn5, Rpn6, Rpn10, and Rpn12) in PA700 (Lid), and seven UPs (Rpn13, Rpt1, Rpt2, Rpt3, Rpt4, Rpt5, and Rpt6) in PA700 (Base) in

*Statistically significant KEGG pathways enriched from 627 UPs. Those UPs were significantly enriched in 47 KEGG pathways (p < 0.05 and FDR < 0.05), among which were included two UPS-related pathways ubiquitin-mediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). The darker dot means the more significant enrichment, and the size of the dot represents the number of UPs enriched in* 

LUSC. No UPs were identified in 20S core particle in LUSC (**Figure 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

#### *Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*

#### **Figure 2.**

*Ubiquitin - Proteasome Pathway*

**Annotation cluster 6**

**Annotation cluster 7**

**Annotation cluster 8**

**Annotation cluster 9**

**Annotation cluster 10**

**Table 2.**

**Category GO term p-value** GOTERM\_CC\_DIRECT Hemoglobin complex 1.97E-02 GOTERM\_MF\_DIRECT Oxygen transporter activity 2.90E-02 GOTERM\_BP\_DIRECT Oxygen transport 3.43E-02

GOTERM\_BP\_DIRECT Viral transcription 1.69E-03 GOTERM\_BP\_DIRECT Translational initiation 5.82E-03 GOTERM\_CC\_DIRECT Ribosome 3.40E-02

GOTERM\_MF\_DIRECT Voltage-gated anion channel activity 5.30E-03 GOTERM\_MF\_DIRECT Porin activity 5.30E-03 GOTERM\_CC\_DIRECT Pore complex 1.11E-02 GOTERM\_BP\_DIRECT Anion transport 3.02E-02 GOTERM\_BP\_DIRECT Regulation of anion transmembrane transport 4.34E-02

GOTERM\_BP\_DIRECT Daunorubicin metabolic process 1.00E-02 GOTERM\_BP\_DIRECT Doxorubicin metabolic process 1.00E-02

GOTERM\_BP\_DIRECT Nucleotide-excision repair, DNA damage recognition 1.00E-02 GOTERM\_BP\_DIRECT Global genome nucleotide-excision repair 2.47E-02

GOTERM\_MF\_DIRECT Neutral amino acid transmembrane transporter activity 1.82E-02 GOTERM\_BP\_DIRECT Neutral amino acid transport 2.62E-02

*The functional categories of 400 DUPs, identified with GO enrichment analysis. Modified from Lu et al. [19],* 

through lysine-63 linkages showed an auto-regulatory mechanism controlling ITCH cytoplasmic-nuclear shuffling [24]. Therefore, the effects of the currently known ubiquitination on these enzymes are only the tip of the iceberg. However, one should also realize that this study found ubiquitination of cullin proteins such as Cul4A and Cul5, while cullin proteins can also be modified by NEDD8 to form NEDDylation. It is well known that the use of the K-ε-GG antibody cannot discriminate between proteins modified with ubiquitin and the related proteins NEDD8 and ISG15. Therefore, for deep investigation of this identified ubiquitination of E3s Cul4A and Cul5 in LUSC in the future, additional experiments are needed to discriminate E3s

Proteasome was a pivotal component for ubiquitin-mediated proteolysis. The 26S proteasome was a complex including two 19S regulatory particles (PA700) and one 20S core particle. The 20S degradation complex contained two α rings (7 subunits, α1-α7) and two β rings (7 subunits, β1-β7). These α rings and β rings together formed a hollow ground circle. The tube-like structure was highly conserved

Cul4A and Cul5 that were modified by ubiquitin, NEDD8, or ISG15 [22].

*with permission from Springer publisher open access article, copyright 2020.*

9.85E-05

5.83E-04

GOTERM\_BP\_DIRECT SRP-dependent cotranslational protein targeting to membrane

GOTERM\_BP\_DIRECT Nuclear-transcribed mRNA catabolic process, nonsensemediated decay

**102**

*Statistically significant KEGG pathways enriched from 627 UPs. Those UPs were significantly enriched in 47 KEGG pathways (p < 0.05 and FDR < 0.05), among which were included two UPS-related pathways ubiquitin-mediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). The darker dot means the more significant enrichment, and the size of the dot represents the number of UPs enriched in the pathway.*

from archaea to mammals [25]. Among them, the α ring was located in the outer layer of the cylinder-like structure, which mainly acted on the recognition of the substrate; the β ring was located in the inner layer of the cylinder-like structure, and was mainly responsible for catalyzing the degradation of the substrate [26, 27]. Three subunits that played a catalytic role were located on the inner surface of the β-ring molecule, exhibiting cysteine protease-like activity, trypsin-like activity, and chymotrypsin-like activity [26, 27]. The 19S regulatory complex contained 19 different subunits, which were divided into two parts: "base" and "lid" [28]. Among them, the base part formed the proximal part of the 19S regulatory complex, which was connected to the alpha ring of the 20S degradation complex, and the lip part formed the distal end. The base section contained 6 ATPase-dependent subunits (Rpt1-Rpt6) and 2 ATPase-independent subunits (Rpn1 and Rpn2) [28]. Usually, Rpn10 and Rpn13 were also classified as the base [26]. The lid part consisted of Rpn3, Rpn5-Rpn9, and Rpn11-Rpn12 subunits [28]. The 19S regulatory complex recognized ubiquitin-labeled target proteins (Rpn10 and Rpn13) and before the target protein entered the 20S degradation complex, deubiquitinated the target protein (Rpn11) and opened the folded structure of the target protein [28]. This study discovered five UPs (Rpn3, Rpn5, Rpn6, Rpn10, and Rpn12) in PA700 (Lid), and seven UPs (Rpn13, Rpt1, Rpt2, Rpt3, Rpt4, Rpt5, and Rpt6) in PA700 (Base) in LUSC. No UPs were identified in 20S core particle in LUSC (**Figure 4**).

#### **Figure 3.**

*UPs identified in ubiquitin-mediated proteolysis pathway (hsa04120). Pink rectangle means the ubiquitinated proteins, and green rectangle means nonubiquitinated proteins. The pathway node in the right panel corresponds to the pink rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. Asterisk (\*) represents there is no quantitative intensity on this modification site in both the tumor and the control group.*


#### **Figure 4.**

*UPs identified in proteasome complex (hsa03050). Pink rectangle means the ubiquitinated subunits, and green rectangle means non-ubiquitinated subunits. The pathway node in the right panel corresponds to the pink rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. T+/N− indicates that this modification site has quantitative intensity only in tumor group, while T−/N+ means this modification site has quantitative intensity only in control group. Asterisk (\*) represents there is no quantitative intensity on this modification site in both the tumor and the control group.*

#### **3.4 DUPs involved in UPS-related molecular network alternations in LUSC**

KEGG pathway network analysis of 400 DUPs revealed 39 statistically significant ubiquitination-mediated signaling pathway alterations (P < 0.05 and FDR < 0.05) (**Figure 5**), including one UPS-related pathway – proteasome complex (hsa03050).

**105**

**Figure 5.**

*article, copyright 2020.*

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

In proteasome complex, this study discovered 4 DUPs (Rpn3, Rpn5, Rpn10, and Rpn6) in PA700 (Lid), and 7 DUPs (Rpn13, and Rpt1-Rpt6) in PA700 (Base). Their ubiquitination levels were significantly increased at residues K74 (Ratio = 5.16) in Rpn10, K34 (Ratio = 2.27) in Rpn13, K293 (T+/N−) in Rpt2, K46 (Ratio = 4.96) in Rpt1, K372 (T+/N−) in Rpt5, K273 (T+/N−) in Rpt4, K346 (T+/N−), K330 (T+/N−) and K290 (T+/N−) in Rpt6, K194 (T+/N−), K328 (T+/N−) and K62 (Ratio = 2.37) in Rpt4, K273 (T+/N−) in Rpn3, K32 (T+/N−) in Rpn6, and K147 (T+/N−) in Rpn5. The ubiquitination level was decreased at residue K53 (Ratio = 0.33) in Rpt5 (**Figure 6**). The proteasome was a pivotal component of UPS to degrade the short-lived regulatory proteins and remove the damaged soluble proteins [29]. Consequently, dysfunction of proteasome might decrease the capability of protein degradation, thus resulting in the increased level of misfolded and damaged proteins, which was closely related to tumorigenesis [30]. The 26S proteasome had one 20S subunit and two 19S regulatory caps. Two 19S caps were necessary to maintain the normal functions of 20S subunit. For example, Rpn 13 in 19S base cap and Rpn 10 in 19S head cap were the recognition-receptors of the ubiquitinated proteins [31, 32]. Further, PTMs (such as phosphorylation, acetylation, myristoylation, and ubiquitination) had been detected in those subunits to greatly complicate the mechanisms of the modulation of proteasome activity. For example, Rpn 10 was mono-ubiquitinated to recruit substrate protein and interact with the shuttle factor of proteasome in drosophila [33, 34]. The multiple ubiquitinations in 19S cap of proteasome such as Rpn 1, Rpn 10, and Rpn 13 were necessary to autophage proteasome [35]. Our study [19] discovered three non-ATPase subunits

*Statistically significant KEGG pathways enriched from 400 DUPs. Those DUPs were significantly enriched in 39 KEGG pathways (p < 0.05 and FDR < 0.05), including one UPS-related pathway – proteasome complex. The darker dot means the more significant enrichment, and the size of the dot represents the number of DUPs enriched in the pathway. Reproduced from Lu et al. [19], with permission from Springer publisher open access* 

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*

#### **Figure 5.**

*Ubiquitin - Proteasome Pathway*

**104**

**Figure 4.**

*in both the tumor and the control group.*

**Figure 3.**

**3.4 DUPs involved in UPS-related molecular network alternations in LUSC**

*UPs identified in proteasome complex (hsa03050). Pink rectangle means the ubiquitinated subunits, and green rectangle means non-ubiquitinated subunits. The pathway node in the right panel corresponds to the pink rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. T+/N− indicates that this modification site has quantitative intensity only in tumor group, while T−/N+ means this modification site has quantitative intensity only in control group. Asterisk (\*) represents there is no quantitative intensity on this modification site* 

*UPs identified in ubiquitin-mediated proteolysis pathway (hsa04120). Pink rectangle means the ubiquitinated proteins, and green rectangle means nonubiquitinated proteins. The pathway node in the right panel corresponds to the pink rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. Asterisk (\*) represents there is no quantitative intensity on this modification site in both the tumor and the control group.*

KEGG pathway network analysis of 400 DUPs revealed 39 statistically significant ubiquitination-mediated signaling pathway alterations (P < 0.05 and FDR < 0.05) (**Figure 5**), including one UPS-related pathway – proteasome complex (hsa03050).

*Statistically significant KEGG pathways enriched from 400 DUPs. Those DUPs were significantly enriched in 39 KEGG pathways (p < 0.05 and FDR < 0.05), including one UPS-related pathway – proteasome complex. The darker dot means the more significant enrichment, and the size of the dot represents the number of DUPs enriched in the pathway. Reproduced from Lu et al. [19], with permission from Springer publisher open access article, copyright 2020.*

In proteasome complex, this study discovered 4 DUPs (Rpn3, Rpn5, Rpn10, and Rpn6) in PA700 (Lid), and 7 DUPs (Rpn13, and Rpt1-Rpt6) in PA700 (Base). Their ubiquitination levels were significantly increased at residues K74 (Ratio = 5.16) in Rpn10, K34 (Ratio = 2.27) in Rpn13, K293 (T+/N−) in Rpt2, K46 (Ratio = 4.96) in Rpt1, K372 (T+/N−) in Rpt5, K273 (T+/N−) in Rpt4, K346 (T+/N−), K330 (T+/N−) and K290 (T+/N−) in Rpt6, K194 (T+/N−), K328 (T+/N−) and K62 (Ratio = 2.37) in Rpt4, K273 (T+/N−) in Rpn3, K32 (T+/N−) in Rpn6, and K147 (T+/N−) in Rpn5. The ubiquitination level was decreased at residue K53 (Ratio = 0.33) in Rpt5 (**Figure 6**).

The proteasome was a pivotal component of UPS to degrade the short-lived regulatory proteins and remove the damaged soluble proteins [29]. Consequently, dysfunction of proteasome might decrease the capability of protein degradation, thus resulting in the increased level of misfolded and damaged proteins, which was closely related to tumorigenesis [30]. The 26S proteasome had one 20S subunit and two 19S regulatory caps. Two 19S caps were necessary to maintain the normal functions of 20S subunit. For example, Rpn 13 in 19S base cap and Rpn 10 in 19S head cap were the recognition-receptors of the ubiquitinated proteins [31, 32]. Further, PTMs (such as phosphorylation, acetylation, myristoylation, and ubiquitination) had been detected in those subunits to greatly complicate the mechanisms of the modulation of proteasome activity. For example, Rpn 10 was mono-ubiquitinated to recruit substrate protein and interact with the shuttle factor of proteasome in drosophila [33, 34]. The multiple ubiquitinations in 19S cap of proteasome such as Rpn 1, Rpn 10, and Rpn 13 were necessary to autophage proteasome [35]. Our study [19] discovered three non-ATPase subunits

#### **Figure 6.**

*DUPs identified in proteasome complex (hsa03050). The red rectangle means the intensities of all identified ubiquitination sites in one protein were increased, and yellow rectangle means at least two ubiquitination sites in a protein with inconsistent ubiquitination intensities. The pathway node in the right panel corresponds to the red and yellow rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. T+/N− indicates that this modification site has quantitative intensity only in tumor group, while T−/N+ means this modification site has quantitative intensity only in control group. Reproduced from Lu et al. [19], with permission from Springer publisher open access article, copyright 2020.*

(PSMD3, PSMD11, and PSMD12), and three ATPase subunits (PSMC1, PSMC4, and PSMC6) were differentially ubiquitinated in 19S regulatory cap of proteasome in LUSC tissues. It clearly demonstrated that these ubiquitinations in 19S regulatory caps might influence the structure and functions of the proteasome complex. Some studies found that PSMD11 was necessary to assemble proteasome complex and elevate the activity of proteasome in embryonic stem cells [36]. Acetylation [37], phosphorylation [38], and SUMO [39] had been reported to occur in PSMD11, and our study first discovered that PSMD11 was ubiquitinated at residue K32 in LUSC tissues but not in control lung tissues [19]. Currently, few literature studies are found regarding the study on the relationship of ubiquitination and function of proteasome subunits. However, the abnormal ubiquitination of proteasome subunits might cause the functional abnormalities of proteasome complex in LUSC tissues and further lead to the imbalance of synthesis and degradation of intracellular proteins. These findings offer the new clues to deeply study and understand the regulation of UPS functions in LUSC.

#### **4. Conclusions**

Label-free quantitative ubiquitinomics was an effective approach to identify ubiquitinated proteins and ubiquitination sites and quantifies the levels of ubiquitination in human LUSC tissues. In total, 627 UPs and 400 DUPs were identified, providing the first (differential) ubiquitinome profile based on fresh human LUSC tissues. GO and KEGG analyses of UPs and DUPs revealed the statistically significant ubiquitination-mediated molecular network alternations, among which several proteins in two UPS-related pathways (ubiquitin-mediated proteolysis pathway, and proteasome complex) underwent ubiquitination in LUSC. Furthermore, 11 subunits

**107**

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas*

of proteasome complex were differentially ubiquitinated in LUSC. These findings demonstrated that ubiquitination was widely present in UPS in LUSC. At the same time, abnormal ubiquitination might affect the functions of the proteasome to promote tumorigenesis and development. This book chapter focused on the status of protein ubiquitination in UPS-related pathways in human LUSC tissues, and provided the scientific data for the elucidation of the specific molecular mechanisms of abnormal ubiquitination during canceration and the development of anti-tumor

The authors acknowledge the financial supports from the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), and the National Natural Science Foundation of

We declare that we have no financial and personal relationships with other

X.Z. conceived the concept, designed the book chapter, wrote and critically revised the book chapter, coordinated, and was responsible for the correspondence

work and financial support. M.L. designed and wrote the book chapter.

DUPs differentially ubiquitinated proteins E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme

KEGG kyoto encyclopedia of genes and genomes

PPPM predictive, preventive and personalized medicine

*DOI: http://dx.doi.org/10.5772/intechopen.93586*

drugs targeting UPS for lung cancer.

China (Grant No. 81572278 to X.Z.).

**Acknowledgements**

**Conflict of interest**

people or organizations.

**Author's contributions**

E3 ubiquitin ligase GO gene ontology

LC liquid chromatography LUSC lung squamous cell carcinoma

LUAD lung adenocarcinoma MS mass spectrometry

UPs ubiquitinated proteins UPS ubiquitin-proteasome system

MS/MS tandem mass spectrometry PTM post-translational modification

**Abbreviations**

*Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas DOI: http://dx.doi.org/10.5772/intechopen.93586*

of proteasome complex were differentially ubiquitinated in LUSC. These findings demonstrated that ubiquitination was widely present in UPS in LUSC. At the same time, abnormal ubiquitination might affect the functions of the proteasome to promote tumorigenesis and development. This book chapter focused on the status of protein ubiquitination in UPS-related pathways in human LUSC tissues, and provided the scientific data for the elucidation of the specific molecular mechanisms of abnormal ubiquitination during canceration and the development of anti-tumor drugs targeting UPS for lung cancer.

#### **Acknowledgements**

*Ubiquitin - Proteasome Pathway*

(PSMD3, PSMD11, and PSMD12), and three ATPase subunits (PSMC1, PSMC4, and PSMC6) were differentially ubiquitinated in 19S regulatory cap of proteasome in LUSC tissues. It clearly demonstrated that these ubiquitinations in 19S regulatory caps might influence the structure and functions of the proteasome complex. Some studies found that PSMD11 was necessary to assemble proteasome complex and elevate the activity of proteasome in embryonic stem cells [36]. Acetylation [37], phosphorylation [38], and SUMO [39] had been reported to occur in PSMD11, and our study first discovered that PSMD11 was ubiquitinated at residue K32 in LUSC tissues but not in control lung tissues [19]. Currently, few literature studies are found regarding the study on the relationship of ubiquitination and function of proteasome subunits. However, the abnormal ubiquitination of proteasome subunits might cause the functional abnormalities of proteasome complex in LUSC tissues and further lead to the imbalance of synthesis and degradation of intracellular proteins. These findings offer the new clues to deeply study

*DUPs identified in proteasome complex (hsa03050). The red rectangle means the intensities of all identified ubiquitination sites in one protein were increased, and yellow rectangle means at least two ubiquitination sites in a protein with inconsistent ubiquitination intensities. The pathway node in the right panel corresponds to the red and yellow rectangle in the left diagram. Protein access number is the Swiss-Prot accession number. Modified sites refer to ubiquitinated lysine (K). Ratio (T/N) = ratio of tumors to controls. T+/N− indicates that this modification site has quantitative intensity only in tumor group, while T−/N+ means this modification site has quantitative intensity only in control group. Reproduced from Lu et al. [19], with permission from Springer* 

Label-free quantitative ubiquitinomics was an effective approach to identify ubiquitinated proteins and ubiquitination sites and quantifies the levels of ubiquitination in human LUSC tissues. In total, 627 UPs and 400 DUPs were identified, providing the first (differential) ubiquitinome profile based on fresh human LUSC tissues. GO and KEGG analyses of UPs and DUPs revealed the statistically significant ubiquitination-mediated molecular network alternations, among which several proteins in two UPS-related pathways (ubiquitin-mediated proteolysis pathway, and proteasome complex) underwent ubiquitination in LUSC. Furthermore, 11 subunits

and understand the regulation of UPS functions in LUSC.

**106**

**4. Conclusions**

**Figure 6.**

*publisher open access article, copyright 2020.*

The authors acknowledge the financial supports from the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), and the National Natural Science Foundation of China (Grant No. 81572278 to X.Z.).

#### **Conflict of interest**

We declare that we have no financial and personal relationships with other people or organizations.

#### **Author's contributions**

X.Z. conceived the concept, designed the book chapter, wrote and critically revised the book chapter, coordinated, and was responsible for the correspondence work and financial support. M.L. designed and wrote the book chapter.

#### **Abbreviations**


*Ubiquitin - Proteasome Pathway*

### **Author details**

Xianquan Zhan1,2,3\* and Miaolong Lu1,2,3

1 University Creative Research Initiatives Center, Shandong First Medical University, Jinan, Shandong, China

2 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan, China

3 State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital, Central South University, Changsha, Hunan, China

\*Address all correspondence to: yjzhan2011@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **References**

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**108**

**Author details**

Xianquan Zhan1,2,3\* and Miaolong Lu1,2,3

University, Jinan, Shandong, China

1 University Creative Research Initiatives Center, Shandong First Medical

Hospital, Central South University, Changsha, Hunan, China

Central South University, Changsha, Hunan, China

provided the original work is properly cited.

\*Address all correspondence to: yjzhan2011@gmail.com

2 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya

3 State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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Vries M, Olsen JV, Vertegaal AC. SUMO-2 orchestrates chromatin modifiers in response to DNA damage. Cell Reports. 2015;**10**:1778-1791

2012;**109**:12449-12454

1994;**269**:7059-7061

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that binds ubiquitin conjugates. The Journal of Biological Chemistry. 1994;**269**:7059-7061

*Ubiquitin - Proteasome Pathway*

hydroxamic acid treatment reveals crosstalks among proteome,

Bernassola F. Itch self-polyubiquitylation occurs through lysine-63 linkages. Biochemical Pharmacology.

[25] Pathare GR, Nagy I, Bohn S, Unverdorben P, Hubert A, Körner R, et al. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proceedings of the National Academy of

Sciences. 2012;**109**:149-154

Series B. 2009;**85**:12-36

Structure. 2002;**10**:609-618

eIF3. Cell. 1998;**94**:615-623

Sciences. 1977;**74**:54-58

2008;**453**:481-488

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[27] Unno M, Mizushima T, Morimoto Y, Tomisugi Y, Tanaka K, Yasuoka N, et al. The structure of the mammalian 20S proteasome at 2.75 Å resolution.

2008;**76**:1515-1521

ubiquitylome and acetylome in nonsmall cell lung cancer A549 cell line. Scientific Reports. 2015;**5**:9520

[18] Qin X, Chen S, Qiu Z, Zhang Y, Qiu F. Proteomic analysis of ubiquitination-associated proteins in a cisplatin-resistant human lung adenocarcinoma cell line. International

Journal of Molecular Medicine.

[19] Lu M, Chen W, Zhuang W, Zhan X. Label-free quantitative identification of abnormally ubiquitinated proteins as useful biomarkers for human lung squamous cell carcinomas. The EPMA

2012;**29**:791-800

Journal. 2020;**11**:73-94

2006;**34**:W720-W724

2011;**20**:214-228

2011;**286**:44403-44411

[24] Scialpi F, Malatesta M, Peschiaroli A, Rossi M, Melino G,

[20] Sherman BT, Tan Q ,

Collins JR, Alvord WG, Roayaei J, Stephens R, et al. The DAVID gene functional classification tool: A novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biology. 2007;**8**:R183

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**110**

[33] Isasa M, Katz EJ, Kim W, Yugo V, González S, Kirkpatrick DS, et al. Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome. Molecular Cell. 2010;**38**:733-745

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[37] Van Damme P, Lasa M, Polevoda B, Gazquez C, Elosegui-Artola A, Kim DS, et al. N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB. Proceedings of the National Academy of Sciences. 2012;**109**:12449-12454

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[39] Hendriks IA, Treffers LW, Verlaan-de Vries M, Olsen JV, Vertegaal AC. SUMO-2 orchestrates chromatin modifiers in response to DNA damage. Cell Reports. 2015;**10**:1778-1791

## *Edited by Xianquan Zhan*

The ubiquitin-proteasome pathway consists of ubiquitin, substrate proteins, E1 enzymes, E2 enzymes, E3 enzymes, and proteasome, which acts in a series of enzymatic reaction chains to ubiquitinate substrate proteins such as surplus and misfolded proteins for degradation by the proteasome to maintain the balance between protein synthesis and degradation in a cell and tissue. Moreover, deubiquitinating enzymes can remove the attached ubiquitin chain, which results in a reverse ubiquitination-deubiquitination process involved in multiple biological processes in a cell and tissue. The changes of components in the ubiquitin-proteasome pathway are associated with multiple pathophysiological processes, such as cancers and neurodegenerative diseases. This book presents the new advances in concepts, analytical methodology, and application of ubiquitin-proteasome pathway for clarification of molecular mechanisms and discovery of new therapeutic targets and drugs in different diseases.

Published in London, UK © 2020 IntechOpen © Dr\_Microbe / iStock

Ubiquitin - Proteasome Pathway

IntechOpen Book Series

Biochemistry, Volume 18

Ubiquitin

Proteasome Pathway

*Edited by Xianquan Zhan*