







































Aresty Rutgers Undergraduate Research Journal, vol. 1, issue 1, Spring 2020 

 
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. 

The Role of Eicosanoids in Regulating the 

Ubiquitin Proteasome System and Proteostasis 
 

Amanpreet Kaur 

 

Abstract 

 

The ubiquitin proteasome system (UPS) is a protein degradation mechanism in eukaryotes 

crucial to maintaining protein homeostasis, or proteostasis. There are tissue-specific 

differences in UPS activity and proteostasis, but the intercellular signalling mechanisms that 

mediate these differences are not well understood. This work examines eicosanoid 

signalling molecules—which are derived from polyunsaturated fatty acids (PUFAs)—and their 

role in proteostasis regulation, particularly the UPS. A reporter transgene that expresses the 

UbG76V-GFP chimeric protein, a metastable substrate for the UPS, is used in Caenorhabditis 

elegans epithelial cells to monitor the level of UPS activity. In wild-type nematodes, 

UbG76V-GFP levels remain high through 24 hours post L4 stage (L4+24). Then, levels 

decrease significantly due to increased UPS activity as the animals age and develop 48 hours 

past L4 (L4+48). Mutants for fat-1, a desaturase enzyme that converts ω-6 PUFAs to ω-3 

PUFAs, exhibited elevated UbG76V-GFP turnover in the hypodermis even at the L4+24 

stage, suggesting that either ω-6 PUFAs (or their eicosanoid derivatives) promote UPS 

activity or ω-3 PUFAs (or their eicosanoid derivatives) inhibit UPS activity. In the intestine, 

mutants for fat-1 showed reduced UbG76V-GFP turnover at the L4+24 and L4+48 life 

stages. Additionally, mutants for emb-8—an NADPH reductase needed to convert PUFAs into 

eicosanoids—also showed reduced UbG76V-GFP turnover in the hypodermis even at the 

L4+48 stage. These results suggest that elements of the eicosanoid signalling pathway, 

including ω-6 PUFAs and their derivatives, significantly contribute to regulation of the UPS 

and proteostasis. 

 

Introduction 

 

In eukaryotic organisms, protein homeostasis involves the cellular control of protein 

synthesis, folding, trafficking, and degradation in order to properly control biological 

processes—specifically, DNA replication, transcription, and mitosis. Rapid cell division 

during these biological processes can cause unfolded or damaged proteins. The initiation of 

refolding these proteins, along with the prevention of protein aggregation, is controlled 

partly by protein degradation mechanisms.  Many human diseases are characterized by an 

inability to degrade damaged or unfolded proteins, particularly those caused by 

neurodegeneration in the brain. The resultant protein aggregation is often responsible for 

the neuronal cell death seen in diseases such as Alzheimer’s, Parkinson’s, and Huntington’s, 

which predominantly affect older populations.   

A major mechanism of protein degradation is the ubiquitin proteasome system (UPS). The 

UPS removes damaged and unfolded target proteins by tagging them with ubiquitin, a small 

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protein, resulting in their recognition and digestion by a large proteasome protease complex 

(Figure 1). In the UPS, an initial ATP hydrolysis step adenylates a molecule of ubiquitin. The 

ubiquitin is then transferred to the target protein to signal for further polyubiquitination. 

Once the chain of ubiquitin molecules on the target protein is at least four units long, that 

substrate becomes a target for degradation by the proteasome. There is a reduction in 

proteostasis that occurs with age, but the specific changes that occur and the pathways that 

affect these changes are currently unclear. 

 

Figure 1. Schematic representation of ubiquitin proteasome system (UPS) activity in the hypodermis. 

Proper UPS function shows that the unstable UbG76V-GFP (green) transgene is quickly polyubiquitinated 

and degraded whereas mRFP (red) remains stable. When not functioning properly, UbG76V-GFP is either 

not polyubiquitinated or not degraded and thus remains stable. Adapted from “Dopamine signaling 

promotes the xenobiotic stress response and protein homeostasis,” by K.K. Joshi, T. L. Matlack, and C. 

Rongo, 2016, The EMBO Journal, 35(17), 1885-1901 (doi: 10.15252/embj.201592524). Copyright 

2016 by EMBO Press. Adapted with permission. 

                                

Cytochrome P450s (CYPs) are reduction-oxidation (redox) enzymes at the end of the 

electron transfer chain.1  Loss-of-function mutants for various CYPs have previously been 

shown to stabilize polyubiquitinated substrates, suggesting decreased UPS activity.2 Certain 

CYPs also metabolize polyunsaturated fatty acids (PUFAs) to eicosanoid signalling molecules 

(Figure 2). PUFAs relevant to eicosanoid signalling can be divided into ω-3 PUFAs, which 

have their first double bond at the third carbon, and ω-6 PUFAs, which have their first 

double bond at carbon 6.  

 

 
1 Lamb et al., 2009 

2 Joshi, Matlack, & Rongo, 2016 



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Figure 2. Schematic representation of ω-6 and ω-3 PUFAs and the CYP-eicosanoid derivative 

signaling pathway. ω-6 PUFAs are converted to ω-3 PUFAs via FAT-1 and PUFA-derived eicosanoids 

are produced via CYPs and EMB-8. 

 

Thus, the eicosanoid synthesis pathway is a worthwhile target for study of UPS regulation. 

Here, various aspects of the eicosanoid synthesis pathway are studied in order to shed light 

on UPS regulation. 

 

Methods 

 

UPS activity can be monitored in Caenorhabditis elegans, a roundworm species, via a 

transgenic approach using the ubiquitin fusion degradation reporter transgene UbG76V-

GFP, which is an unstable protein covalently bonded to a green fluorescent protein (GFP). It 

mimics a monoubiquitinated protein that can be further polyubiquitinated and degraded. 

Once degraded, GFP is no longer detected (Figure 1). Therefore, GFP fluorescence in animals 

expressing UbG76V-GFP is inversely proportional to the level of UPS activity. The UbG76V-

GFP is expressed in epithelial cells from tissue-specific promoters—col-19 in the 

hypodermis and sur-5 in the intestine.  Two red fluorescent proteins, mRFP in the 

hypodermis and mCherry in the intestine, may be monitored alongside UbG76V-GFP and 

used as an internal standard for gene expression. Neither mRFP nor mCherry is 

ubiquitinated, so they do not get degraded. But, they are both expressed from the same 

promoter as GFP, allowing the quantification of gene expression by analyzing the ratios of 

GFP/mRFP and GFP/mCherry (Figure 1). 

 

Essential fatty acids, specifically the ω-3 PUFA alpha-linolenic acid (ALA), are important 

eicosanoid precursors that cannot be synthesized in the human body from 

monounsaturated fatty acids (MUFAs).  However, C. elegans is able to convert other ω-6 

PUFAs to ω-3 PUFAs using the FAT-1 gene, making it a model organism for eicosanoid 

study (Figure 2). Mutants of FAT-1 fail to make ω-3 PUFAs and subsequently accumulate ω-

6 PUFAs. Studying these mutants can help determine whether either type of PUFA distinctly 

contributes to regulating the UPS.  

 



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First, the odIs77 transgene expressing the UbG76V-GFP and mRFP reporters in the 

hypodermis was introduced into a fat-1(wa9) mutant strain and a control odIs77 (wild-type) 

strain (Table 1). C. elegans undergoes four larval stages starting from L1 and ending at L4, 

the final 24-hour larval stage before the worm enters adulthood. A wild-type worm 

expressing UbG76V-GFP and mRFP in the hypodermis quickly accrues both proteins 

between the L4 larval stage and the L4+24 adult stage. Upon entering the L4+48 adult 

stage, which is associated with peak fertility, UbG76V-GFP is swiftly degraded while mRFP 

remains relatively stable (Figure 1).  Both the wild-type and fat-1 strains were imaged at the 

L4+24 and L4+48 stages in order to determine UbG76V-GFP and mRFP levels through 

fluorescence analysis (Table 1). 

 

To establish if UbG76V-GFP turnover with a fat-1 mutant is as quick in the intestine as it is in 

the hypodermis, a fat-1(wa9); psur5 mutant strain homozygous for UbG76V-GFP was 

generated (Table 1). UbG76V-GFP expression in the intestine is less stable than expression in 

the hypodermis, suggesting earlier UPS activity in the intestine. This is possibly due to 

tissue-specific differences in proteasome demand, activity, or expression.  Thus, there was 

a possibility of mistaking typical unstable UbG76V-GFP levels for a heterozygous genotype. 

Furthermore, in the intestine, UbG76V-GFP is on chromosome Ⅲ; the RFP control, mCherry, 

is on chromosome Ⅴ; and the fat-1 mutation is located on chromosome Ⅳ. So, there was a 

possibility of losing either fluorescent tag. Generating the intestinal fat-1 strain in the lab 

allowed greater control over ensuring fluorescence. A pp608 (wild-type) strain, also 

homozygous for UbG76V-GFP and expressing UbG76V-GFP (chromosome III) and mCherry 

(chromosome V) from the sur-5 promoter, was used as a control to determine the impact of 

a fat-1 mutation in the intestine on UPS activity. Both strains were imaged and analyzed at 

the L4+24 and L4+48 life stages (Table 1). 

 

In C. elegans, proper CYP function requires the EMB-8 gene, which codes for a reductase 

enzyme that catalyzes electron transfer from NADPH to the CYP.3 CYPs require EMB-8 to 

metabolize eicosanoids from PUFAs, so emb-8 mutants fail to produce PUFA-derived CYP-

eicosanoids (Figure 2). Studying emb-8 mutants will help characterize the role of EMB-8 

eicosanoid derivatives in UPS regulation.  

 

Because eicosanoids are essential signalling molecules in C. elegans, the emb-8 null 

mutation is a lethal one. As a result, it is difficult to analyze the function of this gene in 

adult animals via a knockout study. To get around this issue, a temperature sensitive allele 

of emb-8 called emb-8(hc69) was synthesized to determine how an emb-8 mutation in the 

hypodermis impacts UPS activity.  Specifically, the emb-8(hc69); odIs76 mutant strain and 

the control odIs76 (wild-type) strain were generated and examined, both with the UbG76V-

GFP and mRFP reporter transgenes again expressed in the hypodermis (Table 1). Both 

strains were imaged and analyzed for fluorescence at the L4+24 and L4+48 stages. The 

L4+72 stage was also imaged for emb-8 mutants to determine the extent of the stability of 

UbG76V-GFP (Table 1). 

 
3 Benenati, Penkov, Muller-Reichert, Entchev, & Kurzchalia, 2009   



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*Only for the emb-8 mutant strain 

 

Table 1. Experimental setup. UbG76V-GFP/mRFP ratios were measured in the hypodermis at 

L4+24 hrs and L4+48 hrs for the fat-1 mutant and odIs77 wild-type strains. UbG76V-

GFP/mRFP ratios were measured in the hypodermis at L4+24 hrs, L4+48 hrs, and L4+72 hrs 

for the emb-8 mutant and pp608 wild-type strains. UbG76V-GFP/mCherry ratios were 

measured in the intestine at L4+24 hrs and L4+48 hrs for the fat-1 mutant and odIs76 

wild-type strains. 

                                   

Results  

 

Accelerated UbG76V-GFP turnover in fat-1 mutants in the hypodermis 

 

As expected, we observed that the wild-type strain exhibited accumulated UbG76V-GFP at the 

L4+24 stage, with similar levels of both UbG76V-GFP and mRFP, giving a mean normalized 

ratio close to 1 (Figures 3 & 5; Table 2). This is consistent with accelerated protein 

accumulation seen at the end of the final larval stage as a wild-type worm enters adulthood, 

likely due to reproduction-associated protein synthesis.4 The wild-type strain exhibited 

reduced UbG76V-GFP levels at the L4+48 stage compared to the internal control mRFP, with a 

mean UbG76V-GFP/mRFP ratio closer to 0 (Figures 7 & 9; Table 2). This is expected in the 

hypodermis of wild-type worms that have reached peak fertility, possibly as a protective 

mechanism against damage caused by protein aggregation.5 The wild-type strain expressed 

mRFP to the same intensity at both stages, which indicates that non-ubiquitinated proteins 

in the hypodermis of wild-type worms are stable (Figures 1, 5, & 9). By contrast, the fat-1 

mutant strain showed increased UbG76V-GFP turnover, showing reduced UbG76V-GFP levels 

and a mean UbG76V-GFP/mRFP ratio close to 0 at both stages (Figures 4 & 8; Table 2). Again, 

in this strain, mRFP remained stable at both stages, which is standard of non-ubiquitinated 

proteins (Figures 6 & 10). 

 

 

 

 

 

 
4 Joshi et al., 2016 

5 Joshi et al., 2016 



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L4+24 hrs                         

odIs77 (WT)                                               fat-1 (wa9); odIs77 

 

 
 

 

L4+48 hrs 

odIs77 (WT)                                             fat-1 (wa9); odIs77 

 

 



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Figures 3-10. UbG76V-GFP turnover is accelerated in the hypodermis of fat-1 mutants. 3 Expression of 

UbG76V-GFP (green) and 5 mRFP (red) from the col-19 promoter at L4+24 hrs in wild-type animals. 4 

Expression of UbG76V-GFP (green) and 6 mRFP (red) from the col-19 promoter at L4+24 hrs in fat-1 

mutants. 7 Expression of UbG76V-GFP (green) and 9 mRFP (red) from the col-19 promoter at L4+48 hrs 

in wild-type animals. 8 Expression of UbG76V-GFP (green) and 10 mRFP (red) from the col-19 promoter 

at L4+48 hrs in fat-1 mutants. 

 

                                                                                                                        

Table 2. fat-1 mutants show accelerated UbG76V-GFP turnover in the hypodermis at L4+24 

hrs. Mean UbG76V-GFP/mRFP ratios at L4+24 hrs and L4+48 hrs in the hypodermis of fat-1 

mutant and wild-type strains. 

                                                                       

At the L4+24 stage, the variation in UbG76V-GFP expression between the wild-type strain and 

the fat-1 mutant strain was significant (p<0.0001) (Figure 11). UbG76V-GFP levels in the 

wild-type strain were noticeably higher than in the fat-1 mutant strain at this stage. At the 

L4+48 stage, both strains expressed low UbG76V-GFP levels (p<0.01) (Figure 12). Taken 

together, these results suggest that the UPS is activated earlier the hypodermis of fat-1 

mutants than in the wild-type strain. As discussed, fat-1 mutants are unable to convert ω-6 

PUFAs to ω-3 PUFAs, which leads to an accumulation of the former and a corresponding 

deficit in the latter (Figure 2). Therefore, there are two mechanisms by which a fat-1 

mutation can cause earlier UPS activation. First, ω-6 PUFAs or their eicosanoid derivatives 

could promote the UPS, so their accumulation would cause premature UPS activity. 

Alternatively, ω-3 PUFAs or their eicosanoid derivatives could inhibit the UPS, so their 

deficiency would accelerate UPS activity.  

 

11                           L4+24 hrs                          12                           L4+48 hrs 

 

Figures 11 and 12. Graphical representation of accelerated UbG76V-GFP turnover in the hypodermis of 

fat-1 mutants. 11 Quantified fluorescence ratios of UbG76V-GFP to mRFP in the hypodermis of 20 



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animals of specified strains at L4+24 hrs. ****p<0.0001. Error bars denote SEM. 12 Quantified 

fluorescence ratios of UbG76V-GFP to mRFP in the hypodermis of 20 animals of specified strains at 

L4+48 hrs. **p<0.01. Error bars denote SEM. 

 

Reduced UbG76V-GFP turnover in fat-1 mutants in the intestine 

 

As predicted in the intestine, the wild-type strain exhibited a mean UbG76V-GFP/mCherry 

ratio close to 0 at both the L4+24 and L4+48 stages (Table 3); UbG76V-GFP fluorescence and 

expression was low in wild-type worms at both stages (Figures 13 & 17). This is consistent 

with the intestinal UbG76V-GFP instability and potentially high underlying UPS activity 

observed in wild-type worms in prior studies.6 As the internal control, mCherry remained 

stable at both stages (Figures 15 & 19). In the fat-1 strain, UbG76V-GFP levels in the intestine 

remained stable (and clearly higher than the wild-type strain) at both the L4+24 and L4+48 

stages with slightly reduced levels at the latter stage (Figures 14 & 18). Once again, the 

mCherry standard remained stable at both stages (Figures 16 & 20). Thus, the fat-1 mutant 

strain displayed a UbG76V-GFP/mCherry ratio close to 1 at the L4+24 stage. This UbG76V-

GFP/mCherry ratio was only moderately reduced at the L4+48 stage, further suggesting 

increased UbG76V-GFP stability in the intestine of fat-1 mutants (Table 3). Given previous 

results with the fat-1 mutant in the hypodermis, it is possible that UPS activity and its 

regulation are subject to tissue-specific differences.  

 

L4+24 hrs 

pp608 (WT)                                              fat-1(wa9); psur-5 

 

 
 

 

 
6 Keith et al., 2016 



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L4+48 hrs 

pp608 (WT)                                              fat-1(wa9); psur-5 

 

 
 

Figures 13-20. UbG76V-GFP turnover is reduced in the intestine of fat-1 mutants. 13 Expression of 

UbG76V-GFP (green) and 15 mCherry (red) from the sur-5 promoter at L4+24 hrs in wild-type animals. 

14 Expression of UbG76V-GFP (green) and 16 mCherry (red) from the sur-5 promoter at L4+24 hrs in 

fat-1 mutants. 17 Expression of UbG76V-GFP (green) and 19 mCherry (red) from the sur-5 promoter at 

L4+48 hrs in wild-type animals. 18 Expression of UbG76V-GFP (green) and 20 mCherry (red) from the 

sur-5 promoter at L4+48 hrs in fat-1 mutants. 

 

 

Table 3. fat-1 mutants show reduced UbG76V-GFP turnover in the intestine at L4+48 hrs. Mean UbG76V-

GFP/mCherry ratios at L4+24 hrs and L4+48 hrs in the intestine of fat-1 mutant and wild-type 

strains.  

 

At both stages, the variation in the UbG76V-GFP fluorescence and expression between the two 

strains was significant (p<0.0001) (Figures 21 & 22). UbG76V-GFP levels in the intestine of 

the wild-type strain were lower compared to the fat-1 mutant strain at both stages. These 

results suggest that, in the intestine of fat-1 mutants, UPS activity is decreased compared to 



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wild-type animals. This contrasts with the hypodermis of fat-1 mutants, which exhibits 

increased UPS activity relative to the wild-type strain. Therefore, PUFAs (or their eicosanoid 

derivatives) in the intestine may have pathways inverse to those in the hypodermis. These 

results are consistent with a mechanism in which either the ω-6 PUFA line inhibits UPS 

activity, or the ω-3 PUFA line promotes UPS activity (Figure 2). Interestingly, wild-type 

worms also appear to differ in UPS activity in the hypodermis and the intestine, with the 

intestine experiencing earlier UPS activity than the hypodermis.7 In total, these results 

suggest that PUFAs or their eicosanoid derivatives regulate UPS activity in a tissue-specific 

fashion. 

 

21                          L4+24 hrs                            22                           L4+48 hrs 

 

Figures 21 and 22. Graphical representation of reduced UbG76V-GFP turnover in the intestine of fat-1 

mutants. 21 Quantified fluorescence ratios of UbG76V-GFP to mCherry in the intestine of 20 animals of 

specified strains at L4+24 hrs. ****p<0.0001. Error bars denote SEM. 22 Quantified fluorescence 

ratios of UbG76V-GFP to mCherry in the intestine of 20 animals of specified strains at L4+48 hrs. 

****p<0.0001. Error bars denote SEM.   

 

Reduced UbG76V-GFP turnover in emb-8 mutants in the hypodermis 

  

In the hypodermis, wild-type C. elegans expressed UbG76V-GFP at elevated levels with a 

mean UbG76V-GFP/mRFP ratio close to 1 at the L4+24 stage, which dropped to near 0 at the 

L4+48 stage (Figures 23 & 27; Table 4). As expected increased protein accumulation was 

seen in the hypodermis of wild-type worms when they first enter adulthood at L4+24 hours, 

and reduced protein levels were seen in the hypodermis of wild-type worms that reached 

peak fertility at L4+48 hours.8 The internal standard, mRFP, remained stable at both stages 

(Figures 25 & 29). Similarly, emb-8 mutants also exhibited accumulated UbG76V-GFP levels at 

the L4+24 stage with a mean UbG76V-GFP/mRFP ratio close to 1. However, emb-8 mutants 

exhibited only slightly reduced UbG76V-GFP levels and UbG76V-GFP/mRFP ratios at the L4+48 

and L4+72 stages (Figures 24, 28, & 31; Table 4). At both of the later stages, UbG76V-GFP 

levels were stable compared to the wild-type strain at the L4+48 stage. Once again, as a 

control, mRFP remained stable in the emb-8 mutant strain at all three stages (Figures 26, 

30, & 32).  

 
7 Keith et al., 2016 

8 Joshi et al., 2016 



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L4+24 hrs 

     odIs76 (WT)                                        emb-8 (hc69); odIs76 

 

 
 

 

 

L4+48 hrs 

    odIs76 (WT)                                        emb-8 (hc69); odIs76 

 

 



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L4+72 hrs: emb-8 (hc69); odIs76 

 

Figures 23-32. UbG76V-GFP turnover is reduced in the hypodermis of emb-8 mutants. 23 Expression of 

UbG76V-GFP (green) and 25 mRFP (red) from the col-19 promoter at L4+24 hrs in wild-type animals. 

24 Expression of UbG76V-GFP (green) and 26 mRFP (red) from the col-19 promoter at L4+24 hrs in 

emb-8 mutants. 27 Expression of UbG76V-GFP (green) and 29 mRFP (red) from the col-19 promoter at 

L4+48 hrs in wild-type animals. 28 Expression of UbG76V-GFP (green) and 30 mRFP (red) from the col-

19 promoter at L4+48 hrs in emb-8 mutants. 31 Expression of UbG76V-GFP (green) and 32 mRFP (red) 

from the col-19 promoter at L4+72 hrs in emb-8 mutants. 

 

 

Table 4. emb-8 mutants show reduced UbG76V-GFP turnover in the hypodermis at L4+24 hrs. Mean 

UbG76V-GFP/mRFP ratios at L4+24 hrs and L4+48 hrs in the hypodermis of emb-8 mutant and wild-

type strains. Mean UbG76V-GFP/mRFP ratio at L4+72 hrs is also shown for the emb-8 mutant strain. 

 

At the L4+24 stage, the variation in UbG76V-GFP fluorescence and expression between the 

wild-type and emb-8 mutant strains was not significant (p>0.05) (Figure 33). These results 

are unsurprising in the hypodermis, given that UbG76V-GFP accumulated at L4+24 hours in 

both strains. And, this aligns with increased protein levels and low UPS activity seen in the 

hypodermis of wild-type animals when they initially enter adulthood.9 However, at the 

L4+48 stage, the difference in UbG76V-GFP fluorescence and expression was highly 

significant (p<0.0001) (Figure 34). Although UbG76V-GFP levels were reduced in the wild-

type strain at L4+48 hours, UbG76V-GFP levels in emb-8 mutants remained stable at L4+48 

hours.10 In the eicosanoid synthesis pathway, the EMB-8 gene is involved in the eventual 

conversion of both ω-6 and ω-3 PUFAs to their eicosanoid derivatives (Figure 2). Thus, if an 

emb-8 mutant stabilizes UbG76V-GFP in the hypodermis by decreasing UPS activity, then 

 
9 Joshi et al., 2016 

10 Joshi et al., 2016 



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these results support an eicosanoid-regulated pathway of the UPS, as opposed to a PUFA-

regulated pathway. It is likely that either ω-6 PUFA-derived eicosanoids promote the UPS or 

ω-3 PUFA-derived eicosanoids repress it. As a whole, these results indicate that UPS activity 

is suppressed in the hypodermis in emb-8 mutants. 

 

                           

33                               L4+24 hrs                       34                           L4+48 hrs 

 

Figures 33 and 34. Graphical representation of reduced UbG76V-GFP turnover in the hypodermis of 

emb-8 mutants. 33 Quantified fluorescence ratios of UbG76V-GFP to mRFP in the hypodermis of 20 

animals of specified strains at L4+24 hrs. Lack of a p-value indicates p>0.05. Error bars denote SEM. 

34 Quantified fluorescence ratios of UbG76V-GFP to mRFP in the hypodermis of 20 animals of specified 

strains at L4+48 hrs. ****p<0.0001. Error bars denote SEM. 

 

Discussion 

  

In order to characterize the role of eicosanoids in the regulation of UPS activity and 

proteostasis, a UbG76V-GFP transgene was introduced into various C. elegans mutants to 

monitor changes in UPS activity, which is inversely related to UbG76V-GFP intensity. In wild-

type C. elegans strains, UbG76V-GFP begins to accrue at the L4+24 stage and is swiftly 

degraded when worms enter peak fertility at the L4+48 stage. In this work, it was confirmed 

that several constituents of the fatty acid-eicosanoid signalling pathway, namely FAT-1 and 

EMB-8, are involved in mediating the UPS. In the hypodermis, fat-1 mutants experienced 

earlier UPS activation relative to wild-type animals, while emb-8 mutants exhibited 

repressed UPS activity. In the intestine, however, fat-1 mutants displayed UPS repression. 

These findings suggest that eicosanoids function in a tissue-specific manner to regulate the 

UPS and maintain proteostasis.  

 

Two mechanisms within the fatty-acid-eicosanoid pathway can be proposed for the 

increased UbG76V-GFP turnover observed in the hypodermis of a fat-1 mutant. Mutants for 

FAT-1 accumulate ω-6 PUFAs because they cannot convert them to ω-3 PUFAs. Therefore, 

one possible explanation is that ω-6 derived CYP-eicosanoids promote UPS activity. This 

means a fat-1 mutation leads to enhanced UPS activity in the hypodermis due to high levels 

of one or more ω-6 PUFAs or their eicosanoid derivatives. Another potential explanation is 

that ω-3 PUFAs or their eicosanoid derivatives inhibit UPS activity, so their absence due to 



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fat-1 results in the de-repression of hypodermal UPS activity. Since the results presented 

here do not suggest one pathway over the other, further studies are required to determine 

which one of these two pathways—if not a combination of both—is responsible for the 

effects of FAT-1 on UPS activity. 

Curiously, fat-1 in the intestine increases UPS activity, which is the opposite of its inhibitory 

role in the hypodermis. Thus, it is likely that eicosanoids exert their effects on the UPS in a 

cell and tissue-specific manner. This is supported by previous studies that found the effects 

of ω-3 PUFAs varied depending on the tissue in question, with reduced UPS activity in 

cachexia cancer cells and increased UPS activity in HPV-infected cancer cells.11 In the 

intestine, ω-3 PUFAs or their derived eicosanoids may promote UPS activity, or ω-6 PUFAs 

or their derived eicosanoids may inhibit it. The exact purpose of tissue-specific 

differentiation in UPS regulation is unknown, although it is possible that there is a greater 

demand to break down proteins in the intestine than in the hypodermis. 

 

Together with CYPs, EMB-8 is responsible for the direct production of eicosanoids. The 

stabilization of UbG76V-GFP resulting from an emb-8 mutation in the hypodermis likely 

suggests a mechanism in which eicosanoids promote UPS activity. This is further supported 

by the observations in the hypodermis of a fat-1 mutant. To determine whether it is ω-6 

PUFAs or ω-6 derived eicosanoids regulating the UPS in the hypodermis, it would be 

worthwhile to study a fat-1 and emb-8 (or even a cyp) double mutant and observe whether 

the emb-8 mutation blocks the increased UbG76V-GFP turnover induced by the fat-1 

mutation. The same experiment could be repeated in the intestine to determine the role of 

eicosanoids in regulating the UPS in a different type of cell. Double mutations in FAT-1 and 

other FAT enzymes would also be valuable in determining the contribution of different ω-6 

PUFAs to UPS regulation and if the effect arises from their derived eicosanoids instead. 

Studying mutations in CEEH-1 and CEEH-2—which convert nonclassical, short-lived 

eicosanoids to classic eicosanoids—would also be helpful in the latter case (Figure 2).  

 

Although the results in this paper have been discussed in the context of eicosanoids 

regulating the UPS, it is also possible that they function through regulating other protein 

degradation mechanisms, such as autophagy or even proteasome activity itself. One study 

observed that supplementing growth media with ω-6 PUFAs activates autophagy in C. 

elegans, which serves to increase starvation resistance and elongate their lifespans.12 

Another publication found that ω-3 PUFAs increased the concentration of ubiquitinated 

proteins in adipocytes, crediting it to reduced expression of proteasome subunits.13 

Although previous studies have examined mutations in possible regulators of the UPS in 

conjunction with direct disruption of the proteasome, the exact role of eicosanoids in 

modulating proteostasis remains unclear.14  

Regardless of their precise function, eicosanoids are key components in eukaryotic 

proteostasis, including protein degradation. Beyond C. elegans, understanding the role of 

 
11 Whitehouse, Smith, Drake, & Tisdale, 2001; Jing et al., 2014 

12 O’Rourke, Kuballa, Xavier, & Ruvkun, 2013 

13 Wójcik et al., 2014 

14 Joshi et al., 2016 



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eicosanoids synthesized from essential fatty acids in proteostasis will help to elucidate their 

biological importance. Improving our understanding of the UPS system may help us to better 

comprehend and prevent age-associated neurodegeneration and disease. 

 

Materials and Procedures 

 

Strains used 

 

The following strains were used: odIs77 (WT)[Pcol-19::UbG76V-GFP, Pcol-19::mRFP], fat-1(wa9); 

odIs77[Pcol-19::UbG76V-GFP, Pcol-19::mRFP], odIs76 (WT)[Pcol-19::UbG76V-GFP, Pcol-19::mRFP]; emb-

8(hc69); odIs76[Pcol-19::UbG76V-GFP, Pcol-19::mRFP], dpy-13[Psur-5::UbG76V-GFP, unc-119(+) Ⅲ, 

Psur-5::mCherry, unc-119(+) V], and hhIs64[Psur-5::UbG76V-GFP, unc-119(+)] Ⅲ; hhIs73[Psur-

5::mCherry, unc-119(+)] V.   

 

Growth conditions 

 

Under standard conditions, worms were grown at 20℃ on nematode growth media (NGM) 

plates seeded with OP50 Escherichia coli. The temperature-sensitive emb-8(hc69); odIs76 

strain was grown at 15℃ until the post-L4 stage, when it was shifted to 20℃, along with the 

wild-type control, odIs76.  

 

To generate the fat-1(wa9); psur-5 strain, the dpy-13; psur-5 strain was used as a balancer 

to introduce psur5 into the fat-1(wa9) strain. 15 hermaphrodites of the dpy-13; psur-5 

strain were first crossed to 20 laboratory N2 (WT) males. Males of the heterozygous F1 

generation were crossed to fat-1(wa9) hermaphrodites. 20 F2 progeny were separated onto 

20 plates and self-crossed. From F3 plates that produced both wild-type and Dpy worms, 

40 phenotypically wild-type worms were separated onto 40 plates and self-crossed. 25 F4 

progeny from F3 plates that produced only phenotypically wild-type progeny were separated 

onto 25 plates. All worms separated displayed UbG76V-GFP and mCherry fluorescence. 

To test if UbG76V-GFP could be observed in the fat-1(wa9); psur-5 strain, 5 F5 progeny from 

each of the 25 F4 plates were separated onto 25 ufd-1 RNAi plates to knock down 

ubiquitination in the worms. Five worms for the odIs77 (WT) strain were also separated onto 

a ufd-1 RNAi plate as a control. Worms from the pp608 (WT) control strain were confirmed 

to be homozygous for UbG76V-GFP by growing on a single ufd-1 RNAi plate. 

 

Statistics 

 

A t-test with Welch’s correction was used in GraphPad Prism to analyze UbG76V-GFP and 

mRFP or mCherry fluorescence values for twenty worms per strain per life stage. Each 

strain’s values were then graphed in GraphPad Prism.  

 

Bleaching and synchronization 

 

Gravid adult worms were harvested with M9 buffer and lysed with modified bleaching 

solution prepared in distilled water containing 10% 10 N NaOH and 10% commercially 



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available bleaching solution. Eggs were resuspended in 20-70 μl of M9 buffer and spotted 

onto NGM plates seeded with E. coli. 

 

Fluorescence microscopy, imaging, and intensity analysis 

 

Twenty worms per strain per life stage were imaged using a 5x objective to detect UbG76V-

GFP, mRFP, and mCherry fluorescence with an epifluorescence microscope and iVision 

software. The worms were first mounted and paralyzed on slides with 2% agarose and 10mM 

tetramisole. The exposure times were 50 ms for L4+24 hrs and 25 ms for L4+48 and L4+72 

hrs. ImageJ was used to outline individual worms in the images and quantify fluorescence. 

Python software was used to calculate mean fluorescence.  

 

RNAi screen 

 

Five individuals from each strain to be screened, including the wild-type control, were put 

on NGM plates with 25 μg/mL carbenicillin and 0.2% lactose. The plates were seeded with E. 

coli that produced dsRNA to knock down expression of UFD-1 and allow assessment of 

UbG76V-GFP homozygosity. A second assessment was conducted by first synchronizing 

worms from each strain through bleaching, including the wild-type control, and spotting the 

eggs onto the RNAi plates. The worms were grown at 20℃ and assessed for UbG76V-GFP 

homozygosity at the L4+24 stage. 

 

Acknowledgements 

 

I am grateful towards Dr. Christopher Rongo for overseeing and supervising this research 

project and for providing valuable suggestions and directions for the research question. I 

would also like to express my gratitude towards Dr. Kishore Joshi for his direct supervision 

of the experiments and for his aid in learning laboratory techniques and background 

information. I also thank Nanci Kane, Mehul Vora, and Nathaly Salazar for their help with 

laboratory equipment and procedures. 

 

References 

 

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Appendix A 

 

Definitions of Terms 

 

ALA. ω-3 PUFA alpha-linolenic acid. Essential fatty acid that cannot be synthesized in the 

human body. Important eicosanoid precursor. 

CEEH-1 and CEEH-2. Catalyze the hydrolysis of short-lived nonclassic eicosanoids to 

eicosanoids. Coded by the ceeh-1 and ceeh-2 genes, respectively. 

C. elegans. Caenorhabditis elegans. Free-living nematodes. Model species for many 

experiments. 

col-19. Promoter from which UbG76V-GFP and mRFP are expressed from in the hypodermis. 

CYP. Cytochrome P450. Oxidation-reduction enzyme at the end of the electron transport 

chain. Facilitates the conversion of PUFAs to eicosanoids. 

DHET and DHEQ. Dihydroxyeicosatrienoic acid and Dihydroxyeicosatrienoic acid. Derived 

from ω-6 PUFAs and ω-3 PUFAs, respectively. Eicosanoid signalling molecules. (Fig 1) 

EET and EEQ. Epoxyeicosatrienoic acid and Epoxyeicosaquatraenoic acid. Derived from ω-6 

PUFAs and ω-3 PUFAs, respectively. Short-lived nonclassic eicosanoid signalling molecules 

converted to eicosanoids by CEEH-1 and CEEH-2. (Fig 1) 

EMB-8. Reductase enzyme required by CYPs to convert PUFAs to eicosanoids. Coded by the 

emb-8 gene. 

emb-8(hc69); odIs76. emb-8 mutant strain used in the hypodermis. Contains a 

temperature-sensitive allele. Typically a lethal mutation. 

FAT-1. Desaturase enzyme that converts ω-6 PUFAs to ω-3 PUFAs. Coded by the fat-1 

gene.  

fat-1 (wa9). fat-1 mutant strain used in the hypodermis. 

fat-1(wa9); psur5. fat-1 mutant strain used in the intestine. 

GFP. Green fluorescent protein. Used as a reporter so that molecules can be imaged and 

analyzed. 

 UbG76V-GFP. Ubiquitin protein, composed of 76 amino acids, with the first being 

glycine and the final being valine, covalently attached to a GFP to construct an  

unstable transgene that can be introduced into research specimens.  

L4 stage. Final larval stage of nematodes. 

 L4+24 stage. First adult stage of nematodes. 24 hours since nematode entered L4. 

 L4+48 stage. 48 hours since nematode entered L4. 

 L4+72 stage. 72 hours since nematode entered L4. 

MUFA. Monounsaturated fatty acid. Fatty acids containing one double bond. Precursors to 

PUFAs. 

odIs76 (WT). Wild-type strain used as the control for the emb-8 mutant in the hypodermis. 

odIs77 (WT). Wild-type strain used as the control for the fat-1 mutant in the hypodermis. 

pp608 (WT). Wild-type strain used as the control for the fat-1 mutant in the intestine. 

PUFA. Polyunsaturated fatty acid. Fatty acids containing more than one double bond. 

Precursors to eicosanoids. 

 ω-3 PUFA. Contains first double bond at carbon 3. Converted from ω-6 PUFAs by  

 the FAT-1 desaturase enzyme. 

 ω-6 PUFA. Contains first double bond at carbon 6. Converted to ω-3 PUFAs by the  



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 FAT-1 desaturase enzyme. 

RFP. Red fluorescent protein. Used as a control reporter so that molecules can be imaged 

and analyzed. 

 mCherry. RFP reporter used in the intestine. 

 mRFP. RFP reporter used in the hypodermis. 

sur-5. Promoter from which UbG76V-GFP and mCherry are expressed from in the intestine. 

UPS. Ubiquitin proteasome system. Mechanism through which damaged or unfolded 

proteins targeted for degradation by the proteasome via ubiquitin tagging. 

 


