Gaucher’s Disease Type 1

Gaucher’s Disease Type 1 is a rare disease that is characterized by an abnormal buildup of
a cellular component called glucosylceramide. Particularly, glucosylceramide can be found in the cell membrane, which is the bilayer that is responsible for protecting and organizing the cell, as well as regulating many important processes such as material exchange with the surroundings. Glucosylceramide itself plays an important role in cell signaling, in which cells coordinate with each other to undergo various functions within the body. Though, the amount of glucosylceramide in cells is regulated by a protein (enzyme) called glucocerebrosidase (GCase). Enzymes are biological catalysts that promote important chemical reactions in the body, and GCase is the one responsible for breaking down glucosylceramide when necessary. In the case of Gaucher’s Disease, this particular enzyme is either absent or non-functional, which in turn results in the accumulation of the component on which it acts (glucosylceramide) and therefore manifestation of Gaucher’s Disease.

From here we must ask, how does GCase become non-functional? And how does the accumulation of glucosylceramide lead to Gaucher’s? When discussing GCase abnormalities, we must first consider what gave rise to GCase. In almost all living organisms, DNA is the hereditary material that is passed through generations and carries the instructions for processes such as growth, reproduction, and all functioning of life. These instructions produce proteins, which are the cellular players that carry out the above processes and the various functions of the body. However, these instructions are sometimes flawed (mutated), which can lead to certain abnormalities. In the case of Gaucher’s Disease, the instructions (GBA1 gene) that give rise to GCase are the ones that are mutated, which results in non-functional GCase that is not able to breakdown glucosylceramide, leading to its buildup.

Now that we established how glucosylceramide accumulates, it is important to ask where it does so. Immune cells called macrophages are rich in small organelles called lysosomes. These lysosomes contain many enzymes that breakdown toxins, and one of the enzymes happens to be GCase. Therefore, buildup of glucosylceramide occurs mainly in macrophages, which are transformed into unique spiral-shaped Gaucher cells not present in any other disease; making them important markers for identification of Gaucher’s Disease.

Over the past several years, research has been done on Gaucher’s in order to identify other factors that may influence its manifestation. In 2016, scientists focused on a previously unrecognized factor that was thought to associate with Gaucher’s disease called PGRN. In some patients, they found that GCase was actually present and functional (which contradicted the traditional hallmark for Gaucher’s), but PGRN was not. Upon further research, they indicated that PGRN was the factor responsible for localizing GCase to lysosomes (where it does its job). Therefore, it turns out that patients could indeed have a normal GCase, but it does not end up where it is functional, which also leads to Gaucher’s. Aside from this, PGRN has another important function that links it to Gaucher’s. It is shown to recruit a repairer (chaperone) of GCase if it does not function properly over time. This provides another role for PGRN in preventing from Gaucher’s and insuring proper GCase activity.

When talking about Gaucher’s Type 1, its symptoms are traditionally associated with enlargement of the liver and spleen. Recently however, Gaucher’s has been also associated with inflammation (swelling, redness) – though the mechanism for this had not been understood. In 2015, a study illustrated that one of the two products (ceramide) of the breakdown of glucosylceramide by GCase suppresses a factor that induces inflammation. Therefore, when GCase is non-functional in Gaucher’s Disease, not only is there excess glucosylceramide, but also a deficiency in the products of glucosylceramide breakdown. This in turn promotes inflammation, and supports the characterization of Gaucher’s as an inflammatory disease. 

Recent studies have also increasingly linked Gaucher’s Disease to other famous diseases such as Parkinson’s Disease. This disease is characterized by stiffness and slowing of movement, typically more prevalent with age. In 2016, a study proposed a mechanism in which non-functional GCase is directly linked to symptoms of Parkinson’s disease. Conversely, when GCase is restored, these symptoms are relieved. Ultimately, it was found that GCase processes a main factor that causes Parkinson’s and prohibits it from taking place, therefore preventing individuals from the disease.

Fortunately, there has been a couple ways to treat Gaucher’s. The first treatment (called Substrate Reduction Therapy) is to prevent glucosylceramide from accumulating by targeting its production. The second is called Enzyme Repacement Therapy, which is accomplished by taking doses of functional GCase. Though, these both have many side effects and are not effective against the associated diseases discussed. Thus, the field is currently focusing on newer therapies that introduce repairers (chaperones) of both existing GCase and factors not only linked to Gaucher’s, but other diseases like Parkinson’s as well.

For further information, please visit my Theme Pages and access the PDF version of the infographic above Gaucher’s Disease Infographic.

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A Shared 3D Conformation Between Alzheimer, Parkinson and Huntington Oligomers Impairs Ubiquitin-Dependent Proteasome Function

Opposing allosteric controls between two allosteric modulators-HbYX motif and A11 oligomers- that bind to distinct sites on the 20S proteasome.

Name of paper: “A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers”



From our discussions regarding ubiquitation in BCM441 a couple weeks ago, we began to understand its importance for the degradation of misfolded proteins, along with its role in the removal of aggregates and damaged organelles through processes such as autophagy and mitophagy. We also discussed the role of E3 ubiquitin-protein ligase (Parkin) mutations in disrupting this necessary ubiquitination process, which led to the accumulation of mitochondrial damage and protein aggregates directly linked to neurodegenerative diseases such as Parkinson’s. Therefore, these discussions, along with previous studies on ubiquitation, reason that neurodegenerative diseases mainly result from the idea that proteins that normally should be degraded do not do so after misfolding occurs, causing neuronal pathologies. Moreover, it has been demonstrated that the  aggregated proteins themselves become capable of interacting and interfering with proteasomes, which in turn further impairs ubiquitation and contributes to these pathologies (Bence et al., 2001). Though, despite these findings, it has not been demonstrated what types of aggregates specifically impair proteasome function, and a canonical understanding of why and how proteasomes are widely disrupted in neurodegenerative diseases has not been established (Thibaudeau et al., 2018). As a result,  Thibaudeau et al. attempt to address these issues and introduce a mechanism that characterizes proteasome dysfunction caused by misfolded oligomers in Parkinson, Alzheimer and Huntington neurodegenerative diseases.


Ubiquitinated proteins are recognized for degradation by the 26S proteasome (Thibaudeau et al., 2018). It is made up of a 20S core particle capped on one or both ends by the 19S ATPase regulatory particle (Thibaudeau et al., 2018). The multistep process in which proteins get degraded starts with the 19S regulatory particle binding ubiquitinated substrates and opening a substrate entry gate in the 20S (Thibaudeau et al., 2018). It then unfolds the substrates by translocating them into a 20S catalytic chamber composed of four heteroheptameric rings arranged in a α7-β7-β7-α7 fashion where they get degraded (Groll et al., 2000). Most notably, the opening of the gate requires the C-terminal HbYX motif of the 19S ATPases to bind on top of the 20S, which alludes to the importance of 20S proteasome gate regulation for proper functionality (Thibaudeau et al., 2018). Therefore, the authors hypothesize that toxic oligomers specifically impair HbYX motif-dependent gate opening through a shared mechanism, which in turn blocks protein degradation and disrupts proteasome functionality.


Considering the inconsistency in the literature regarding the effects of aggregates on proteasome functionality, Thibaudeau et al. begin by generating populations of protein aggregates from either amyloid-β 1–42 (Aβ), α-Syn, or huntingtin exon 1 with a polyQ-expansion (Htt-53Q) (which correspond to those found in Alzheimer’s, Parkinson’s and Huntington’s respectively) and observed their impacts on proteasome functionality. Indeed, they found that the 20S proteasome ability to hydrolyze fluorogenic peptide substrates was impaired (Thibaudeau et al., 2018). Moreover, upon separation of soluble oligomers from insoluble ones, the authors found that only the former had an impact on proteasome function (in a concentration-depedent manner) (Thibaudeau et al., 2018). Considering this finding, along with the fact that Aβ, α-Syn, and Htt-53Q monomers were in fact degraded by the proteasomes, the authors inferred that proteasome impairment might be due to the structure of the soluble oligomers. Therefore, the authors replicated the generation of a poly-clonal anti-oligomer antibody (A11) that specifically recognizes some types of protein oligomers regardless of primary structure, initially developed by Kayed et al. Following the incubation of these antibodies with the oligomers, they were surprised that all three soluble oligomer types displayed strong A11 antibody binding (Thibaudeau et al., 2018), which hints to structural similarity between the oligomers.


The authors then went on to address whether proteasome impairment was due to oligomer size rather than structure and if shared A11 reactivity was merely a coincidence (Thibaudeau et al., 2018). Thus, they generated both 200–400 kDa A11+ Aβ oligomers and larger 700 kDa ones, and found that both retained the ability to impair substrate hydrolysis by all three active sites of the proteasome but to a lesser degree by the latter. This was expected  based on results from a previous study that demonstrate how protofibrils (elongated clusters of cells that grow into fibrils) form when oligomers bind to one another in forming a chain of oligomers, which sterically block internal oligomers but not terminal ones from interacting with the proteasome (Yang et al., 2017). Before moving on, the authors also wanted to check whether the structural epitope of the A11 antibody on the Aβ oligomer was necessary for proteasome inhibition (Thibaudeau et al., 2018). And indeed, when the antibody was bound to the oligomers, proteasome function was rescued; however, when an antibody was bound to the N-terminus instead (as a control), functionality was disrupted. These results suggested that a specific A11 epitope site is essential for proteasome dysfunction induced by the soluble oligomers (Thibaudeau et al., 2018).


In order to characterize the mechanism of proteasome impairment, the authors adopt an established protocol from Barghorn et al. to generate physiologically relevant Aβ*56 oligomers in which the A11+ epitope was maintained (consistent with those purified from human brain tissue in Alzheimer’s) (Thibaudeau et al., 2018). The authors then observed whether these oligomers could directly interact with the 20S proteasome. They found that the oligomers were clearly co-migrating with the 20S proteasome visualized by Native-PAGE gel (Thibaudeau et al., 2018). Therefore, this confirmed that these oligomers physically interact with the proteasome which in turn leads to its dysfunction.


From here, the authors went on to address whether the impaired 20S proteasome function was due to impaired substrate entry through the gate or by allosterically impairing the active sites (Thibaudeau et al., 2018). To do so, they compared the impacts of the three A11+ oligomers (Aβ*56, α-Syn, and Htt-53Q) on WT proteasome and mutant proteasome α3ΔN  which has a constitutively open gate. They found that only the WT proteasome was impaired, but not the mutant; suggesting that the oligomers require a functioning gate to impair proteasome activity (Thibaudeau et al., 2018). Further, they found that all three catalytic sites were impaired in the WT as opposed to only one or two for example, which also contributes to the suggestion that substrate access altogether might be inhibited. In addition, the authors perform a substrate saturation curve on the WT proteasome with and without the Aβ*56 oligomers. Through the use of nonlinear regression and Michaelis–Menten kinetic analysis, they found that the oligomers resulted in a decrease in Vmax and an increase in Km, which are characteristic of allosteric (non-active site) inhibition. Thus, they suggested that all three oligomers impair the proteasome with the same allosteric mechanism, since they all require the closable gate to impair function (Thibaudeau et al., 2018).


As previously mentioned, the opening of the 20S proteasome gate requires the C-terminal HbYX motif of the 19S ATPases to bind on top of the 20S, which induces a conformational change of the 20S α- subunits and stabilizes the open state of the N-terminal gating residues (Thibaudeau et al., 2018). However, the 11S family of proteasome activators (PA28α/β and PA26) lack the HbYX motif of the 19S ATPases and open the gate through an 11S activation loop, which is independent of the HbYX mechanism (Whitby et al., 2000). Thus, the authors elected to test which mechanism of these two is the one impaired by the disease-related oligomers (Thibaudeau et al., 2018). Upon addition of the A11 oligomers with saturating amounts of PA26 activator in one trial and PA28α/β in another, they found that  none of the oligomers were able to impair proteasome function in either case. These results were further confirmed via kinetic analysis, suggesting that the A11 oligomers bind to the 20S proteasome at a location separate from the 11S proteasome activators (Thibaudeau et al., 2018).


Next, the authors tested if A11+ Aβ*56 oligomers could impair ubiquitin-dependent (Ub4(lin)-GFP-35) protein degradation by purified human 26S proteasomes (Thibaudeau et al., 2018). Indeed, they found that protein degradation was severely impaired. Considering that this process requires ATP-dependent unfolding and injection into the 20S core, this data suggested that the oligomers impair the HbYX mechanism of gate opening (Thibaudeau et al., 2018). Through native-PAGE analysis, the authors confirm that this impairment was not due to dissociation of the 19S and 20S, further strengthening their results of an allosteric (and not competitive) inhibition. To further validate their results, the authors prepared mouse brain lysates that resemble the complex heterogeneous cellular environment in vivo. Indeed, the oligomers nearly completely impaired proteasome function even in this complex environment (Thibaudeau et al., 2018).


Considering that the 26S proteasome adopts multiple conformations during the ATP hydrolysis cycle by 19S ATPase, the authors examine the impact of the oligomers on these conformations. It is known that hydrolysable ATP stabilizes the closed gate conformation, while non-hydrolyzing ATP analogues better stabilize the open gate form (Thibaudeau et al., 2018). Surprisingly, the oligomers impaired the normal physiological (with ATP) state of the 26S but not the synthetically opened state (using ATPγS analog). These results support the inhibition of the HbYX-dependent conformational changes that lead to gate opening, as opposed to inhibition of the proteasome after the gate is opened (Thibaudeau et al., 2018). Moreover, the authors tested this hypothesis by adding the A11 oligomers in the presence and absence of an established gate opening peptide Rpt5. The authors found that the oligomers did impair gate opening by Rpt5; however, they were not as effective when high concentrations of Rpt5 were added (1mM). These confirm the impairment of HbYX-dependent gate opening, but what was also important is that the HbYX peptide could overcome impairment by the oligomers at higher concentrations (Thibaudeau et al., 2018).


To summarize, the authors propose the following mechanistic model of how A11 oligomers inhibit proteasome function: They bind to the outer surface of the proteasome (specifically the α-subunits along the C2 axis); this in turn leads to stabilization of the closed-gate conformation and prevention of HbYX-dependent gate opening (as opposed to no impairment of gate opening by the 11S proteasome activators). The model proposes opposing allosteric controls between two allosteric modulators (the HbYX motif and the oligomers) that bind to distinct sites on the 20S proteasome, with the motif having an activating effect on the proteasome vs. the oligomers having an impairing effect (Thibaudeau et al., 2018). Ultimately, the study demonstrates that proteasome impairment achieved by Alzheimer, Parkinson and Huntington oligomers are due to 3D shape, as opposed to primary structure and sequence.


Overall, the study is very important in that it proposes a mechanism in which soluble aggregates from three very dangerous diseases all share a common way to impair proteasome function. It is also high yield in the sense that results were consistent when mimicking physiological conditions. However, many questions remain with regard to future studies and therapeutic applications. For example, what levels of physiological oligomers are sufficient to impair activity? Also, considering that the HbYX motif and non-hydrolyzing ATP stabilize the open-gate form, would it be possible to prevent the hydrolysis of ATP to ADP and thus favor the open-gate form and retain normal function? If these questions are addressed, translational interventions may have the potential to restore normal degradation of misfolded proteins, and decrease the severity of the neurodegenerative diseases discussed in this study.





  • Barghorn, Stefan, Volker Nimmrich, Andreas Striebinger, Carsten Krantz, Patrick Keller, Bodo Janson, Michael Bahr, et al. 2005. “Globular Amyloid Beta-Peptide Oligomer – a Homogenous and Stable Neuropathological Protein in Alzheimer’s Disease.” Journal of Neurochemistry 95 (3): 834–47.
  • Bence, N. F., R. M. Sampat, and R. R. Kopito. 2001. “Impairment of the Ubiquitin-Proteasome System by Protein Aggregation.” Science (New York, N.Y.) 292 (5521): 1552–55.
  • Groll, Michael, Monica Bajorek, Alwin Köhler, Luis Moroder, David M. Rubin, Robert Huber, Michael H. Glickman, and Daniel Finley. 2000. “A Gated Channel into the Proteasome Core Particle.” Nature Structural & Molecular Biology 7 (11): 1062–67.
  • Kayed, Rakez, Elizabeth Head, Jennifer L. Thompson, Theresa M. McIntire, Saskia C. Milton, Carl W. Cotman, and Charles G. Glabe. 2003. “Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis.” Science (New York, N.Y.) 300 (5618): 486–89.
  • Thibaudeau, Tiffany A., Raymond T. Anderson, and David M. Smith. 2018. “A Common Mechanism of Proteasome Impairment by Neurodegenerative Disease-Associated Oligomers.” Nature Communications 9 (1): 1097.
  • Whitby, F. G., E. I. Masters, L. Kramer, J. R. Knowlton, Y. Yao, C. C. Wang, and C. P. Hill. 2000. “Structural Basis for the Activation of 20S Proteasomes by 11S Regulators.” Nature 408 (6808): 115–20.
  • Yang, Ting, Shaomin Li, Huixin Xu, Dominic M. Walsh, and Dennis J. Selkoe. 2017. “Large Soluble Oligomers of Amyloid β-Protein from Alzheimer Brain Are Far Less Neuroactive Than the Smaller Oligomers to Which They Dissociate.” The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 37 (1): 152–63.
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Reflection Blog 3

In collecting the references for my project on Gaucher’s disease, I began by exploring literature that defines what the disease is and looked up papers that discuss its history and  prevalence over the years. This was labeled under “Historical Overview of Gaucher’s Disease”. My next theme was titled “Structural Biology of Gaucher’s Disease”. Here, I searched for papers that characterize the structure of the malfunctioning enzyme and define the residues that comprise the active site and other important domains. This would lead to my third project theme, titled “Disease Mechanism/Pathophysiology”. Here, I collected papers that identify the various mutations that lead to the disease, and how each mutation impacts the structure of the enzyme in its unique way. Considering the association of Gaucher’s disease to various other diseases and pathways, I researched how the buildup of the substrate leads to different abnormalities depending on the cellular location in which this occurs, and how the misfolding of the enzyme in Gaucher’s can interfere with physiological functions and increase the risk of developing other pathologies. This led to my next project theme, “Related Diseases”. Under this theme, I collected papers that dissect the biochemical relationship between Gaucher’s disease and diseases such as Parkinson’s, myeloma and hyperferritinemia.  From here, I looked into some therapies that have been traditionally utilized in the field (such as substrate reduction therapy and enzyme replacement therapy) and labeled those under the theme “Traditional Therapies”. As a result, this also led me to consider searching for newer therapies that not only focus on Gaucher’s disease itself, but also have the potential to alleviate the newly associated symptoms of Parkinson’s and myeloma (“Improved Therapies”). One such therapy is through the use of newly developed pharmacological chaperones, which provide stronger evidence as potential treatments against all of the mentioned diseases. Lastly, I explored some challenges that still exist in the field today, and potential issues that need to be addressed in the near future (“Future Work/Major Challenges”). These include attempting to understand the different organ involvement in Gaucher’s disease; determining the relative efficiencies of different treatments; and resolving the dilemma in whether there truly exists both a non-neuropathic (Type 1) and a neuropathic form of Gaucher’s (Types 2 and 3), considering that both are associated with Parkinson’s disease, as opposed to the latter form only. In conclusion, the chosen organization of these themes allows me to: 1) address the disease from a historical perspective; 2) demonstrate what has been recently discovered regarding structure and pathologies; and 3) what innovations move the field forward and some challenges that are important to address in the near future.

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Omega-3 Docosahexaenoic Fatty Acid Kills Breast Cancer Cells: Unraveling the Complex Pathway that Triggers Cancer Apoptosis Induced by N-3 DHA PolyUnsaturated Fatty Acid

Scheme summarizing the DHA induction of OSGIN1-mediated the onset of apoptosis via the up-regulation of PI3K/Akt/Nrf2 signaling pathway in breast cancer cells


Name of paper: Docosahexaenoic acid (DHA) increases the expression of oxidative stress- induced growth inhibitor 1 through the PI3K/Akt/Nrf2 signaling pathway in breast cancer cells




From our discussions about lipids in BCM 441 over the past few weeks, we started to gain more insights on the different classifications of lipids, especially with regard to poly-unsaturated fatty acids (PUFAs). We learned that these lipids can be categorized based on the position of the first double bond from the methyl end of the fatty acid chain, with each of the omega-3 (N-3) double bond and omega-6 (N-6) double bond being located between the third and fourth carbon, and the sixth and seventh carbon, respectively. Particularly, omega-3 α-linolenic and omega-6 linoleic are essential fatty acids to humans, as they must be acquired from different plant species in order to extend and functionalize them to different metabolites. These fatty acids are extremely beneficial, since they have been linked to improved cardiovascular health, along with decreased risks of depression, Alzheimer’s disease and cancer (Shahidi and Ambigaipalan, 2018). For example, a recent study illustrated that omega-3 eicosapentaenoic and docosahexaenoic acids (EPA and DHA), along with omega-6 gamma-linolenic and arachidonic acids (GLA and AA) all induced apoptosis in the colon cancer cells LoVo and RKO (Zhang et al., 2015). Despite these benefits however, the mechanism in which fatty acids might prevent different cancers from developing has not been fully understood or characterized (Chia-Han et al., 2017). Therefore, in this paper, Chia-Han et al. attempt to both characterize how PUFAs might inhibit the proliferation of breast cancer cells, and develop a full pathway that describes how DHA can lead to the apoptosis of breast cancer tumors.


Oxidative stress-induced growth inhibitor 1 OKL38 (or OSGIN1) is a primary tumor inhibitor gene that results in the apoptosis of many carcinoma cells upon its expression (Li et al., 2007). Expression is mediated via the activity of transcription factor nuclear factor E2-related factor (Nrf2), which is a common factor involved in the regulation of many genes that are responsive to oxidative stress in the cell (Li et al., 2007). Under these conditions, Nrf2 translocates from the cytosol to the cell nucleus and forms a heterodimer with small Maf proteins (sMafs) in order to bind to antioxidant response element promoters (AREs) (Kocanova et al., 2007; Li et al., 2008). Studies have also demonstrated that Nrf2 silencing with siRNA significantly reduced stimulated expression of OKL38 in human aortic endothelial cells and thus promoted cancer (Yan et al., 2014). Other players such as extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase/Akt (PI3K/ Akt) are also involved in Nrf2-mediated gene transactivation, but their roles have not been fully elucidated (Kocanova et al., 2007).


Therefore, considering that N-3 and N-6 PUFAs have been linked to the up-regulation of certain apoptosis-related proteins in cancer cells, along with the centrality of OKL38 as a predictive biomarker for tumorigenesis, Chia-Han et al. elected to study the potential of PUFAs to induce the expression of OKL38, and identify a plausible mechanism that results in cancer cell death. Further, considering that breast cancer is one of the most prevalent cancers worldwide, especially among African-American women and other minorities (Desantis et al. 2016), the authors devote their research to MCF-7 and Hs578T human breast cancer cells, and compare the differences to non-tumorigenic mammary epithelial cells (Chia-Han et al., 2017).


The authors first assayed the effect of 100 uM AA, LA, GLA, ALA, EPA, and DHA fatty acids on OKL38 expression in both MCF-7 and Hs578T cancer cells, and found that only DHA significantly up-regulated its expression (Chia-Han et al., 2017). They also found that expression was dose-dependent, as lower concentrations of DHA led to decreased expression. They also found that increased concentrations of the other PUFAs had no impact on OKL38 expression. Not only does this distinguish between omega-3 and omega-6 functionalities on OKL38, but also indicates that only omega-3 DHA impacts expression and not omega-3 EPA, which was not expected (Chia-Han et al., 2017). The authors then went on to assess the effect of DHA on the nuclear translocation of Nrf2; and indeed, they demonstrated higher levels of Nrf2 accumulation in the nuclear fraction upon addition of DHA. To verify the role of Nrf2 in DHA-induced OKL38 expression, the authors found that silencing Nrf2 significantly reduced OKL38 expression despite addition of DHA. This suggests that up-regulation of Nrf2 expression and nuclear translocation are critical for the induction of OKL38 by DHA in breast cancer cells (Chia-Han et al., 2017).


To further investigate the role of other components in DHA-induced OKL38 expression, the authors also inhibited MAPKs and PI3K/Akt signaling pathways that were shown to up-regulate Nrf2 expression from previous studies (Yang et al., 2013). They found that both DHA-induced OKL38 protein expression and Nrf2 translocation were significantly inhibited by the PI3K inhibitor (Chia-Han et al., 2017). These findings extend the previous results, and suggest that PI3K/Akt signaling also plays a major role in Nrf2-mediated OKL38 expression by DHA (Chia-Han et al., 2017).


Considering that the PI3K/Akt signaling pathway is mainly one that is responsive to oxidative stress (Li et al., 2007), Chia-Han et al. elected to test the hypothesis that up-regulation of OKL38 by DHA occurs through the induction of Reactive Oxygen Species (ROS). Indeed, they found an increase in ROS after treating breast cancer cells with DHA. Further, upon adding a ROS scavenger, OKL38 expression was significantly reduced, and translocation of Nrf2 was significantly attenuated, suggesting that DHA deliberately increases ROS in cells, which activates the pathway that leads to tumor suppression.


After characterizing the specific function of DHA in inducing breast cancer apoptosis, the authors elected to identify the entire pathway that leads to cancer death. With previous knowledge of proteins that have demonstrated their ability to reduce cancer, such as BCL2 family proteins, p53, caspases, and cytochrome c (Kwon et al., 2008), the authors compared the expression of these proteins in DHA-treated cancer cells to non-DHA treated cells (Chia-Han et al., 2017). They found that Bcl-2 expression decreased while Bax increased in DHA-treated cells. Further, p53 significantly accumulated in the mitochondria, and cytochrome c was notably released from the mitochondria to the cytosol, which initiated cell apoptosis. Therefore, the authors create a scheme that summarizes the apoptosis pathway by DHA induction of OKL38 (refer to cover image). As a summary, the pathway begins with increased ROS induced by DHA, which leads to the activation of PI3K/Akt and translocation of Nrf2 from the cytosol to the nucleus. This then leads to up-regulation of the tumor suppressing protein OKL38, which results in both the increase of the Bax/Bcl-2 ratio and translocation of p53 to the mitochondria. p53 then induces the release of cytochrome c from the mitochondria, which results in the death of the breast cancer cell.


Finally, the authors sought to confirm whether the proapoptotic effects of DHA were observed only in breast cancer cells (Chia-Han et al., 2017). They compared MCF-7 breast cancer cells with chemically transformed human mammary epithelial cells (H184) and non-tumorigenic epithelial cells (MCF-10A). They found that treatment with 100 and 200 uM of DHA increased apoptosis in MCF7 cells, but not in MCF-10A. However, as the concentration of DHA increased, more death was observed in MCF-10A. These results suggest that high levels of DHA lead to the death of both breast cancer cells along with normal mammary epithelial cells. Furthermore, accumulation of p53 in the mitochondria and cytochrome c release was significantly higher in MCF7 cells than in both H184 and MCF-10A when induced with 100 uM DHA only. All of these results combined provide evidence for preferential DHA-induced apoptosis in breast cancer cells as opposed to nontumorigenic breast epithelial cells (Chia-Han et al., 2017).


This is a tremendous study that elucidates how consuming an omega-3 rich diet, particularly DHA, can potentially prevent from breast cancer. The study has high-impact qualities, considering the characterization of a new apoptosis pathway induced by DHA (and thus ROS ironically), along with the application to both nontumorigenic and cancer breast cells. Other perils from this study are resembled in its potential translational applications in the future to help combat breast cancer. For example, considering that 100 uM DHA triggered apoptosis in cancerous cells but not in normal cells, specific doses can be administered in chemotherapy to help kill cancer cells but cause no harm to the normal cells. Simultaneously, since higher concentrations killed both types of cells, precautions can be established in future clinical therapies in order to provide more efficient and less dangerous treatments. Finally, this study shows that even an excess of omega-3 fats can cause serious health problems. Considering that most studies focus on the harm of excess omega-6 fats and leave out the dangers of excessive omega-3 fat consumption, this study becomes unique in its illustration that DHA can indeed reduce cancer, but also kill normal cells if not consumed in moderation.



  1. Tsai, Chia-Han, You-Cheng Shen, Haw-Wen Chen, Kai-Li Liu, Jer-Wei Chang, Pei-Yin Chen, Chen-Yu Lin, Hsien-Tsung Yao, and Chien-Chun Li. 2017. “Docosahexaenoic Acid Increases the Expression of Oxidative Stress-Induced Growth Inhibitor 1 through the PI3K/Akt/Nrf2 Signaling Pathway in Breast Cancer Cells.” Food and Chemical Toxicology 108 (October): 276–88.
  2. Li, Rongsong, Wendy Chen, Rolando Yanes, Sangderk Lee, and Judith A. Berliner. 2007. “OKL38 Is an Oxidative Stress Response Gene Stimulated by Oxidized Phospholipids.” Journal of Lipid Research 48 (3): 709–15.
  3. Kocanova, Silvia, Esther Buytaert, Jean-Yves Matroule, Jacques Piette, Jakub Golab, Peter de Witte, and Patrizia Agostinis. 2007. “Induction of Heme-Oxygenase 1 Requires the P38<Superscript>MAPK</Superscript> and PI3K Pathways and Suppresses Apoptotic Cell Death Following Hypericin-Mediated Photodynamic Therapy.” Apoptosis 12 (4): 731–41.
  4. Li, Wenge, Siwang Yu, Tong Liu, Jung-Hwan Kim, Volker Blank, Hong Li, and A. -N. Tony Kong. 2008. “Heterodimerization with Small Maf Proteins Enhances Nuclear Retention of Nrf2 via Masking the NESzip Motif.” Biochimica et Biophysica Acta (BBA) – Molecular Cell Research 1783 (10): 1847–56.
  5. DeSantis, Carol E., Rebecca L. Siegel, Ann Goding Sauer, Kimberly D. Miller, Stacey A. Fedewa, Kassandra I. Alcaraz, and Ahmedin Jemal. 2016. “Cancer Statistics for African Americans, 2016: Progress and Opportunities in Reducing Racial Disparities.” CA: A Cancer Journal for Clinicians 66 (4): 290–308.
  6. Yan, Xinmin, Sangderk Lee, B. Gabriel Gugiu, Lukasz Koroniak, Michael E. Jung, Judith Berliner, Jinluo Cheng, and Rongsong Li. 2014. “Fatty Acid Epoxyisoprostane E2 Stimulates an Oxidative Stress Response in Endothelial Cells.” Biochemical and Biophysical Research Communications 444 (1): 69–74.
  7. Kwon, Jae Im, Gi-Young Kim, Kun Young Park, Chung Ho Ryu, and Yung Hyun Choi. 2008. “Induction of Apoptosis by Linoleic Acid Is Associated with the Modulation of Bcl-2 Family and Fas/FasL System and Activation of Caspases in AGS Human Gastric Adenocarcinoma Cells.” Journal of Medicinal Food 11 (1): 1–8.
  8. Zhang, C., Yu, H., Shen, Y., Ni, X., et al., 2015. Polyunsaturated fatty acids trigger apoptosis of colon cancer cells through a mitochondrial pathway. Arch. Med. Sci. 11, 1081-1094.


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Reflection Blog 2

One of the many perils of studying biochemistry is that we get to navigate the complexity of life from both “bottom-up” and “top-down” perspectives. We no longer become content with learning “what” things are, but rather strive to understand how things occur in biological systems at the chemical and molecular levels. Personally, I have always found this gratifying, especially when it comes to learning about infectious diseases in humans. In particular, I had always been intrigued by the complexity of certain diseases, and have strived to learn more about them.


The first disease that I am concerned about is Parkinson’s disease. This progressive disease is characterized by gradual loss in mobility and muscle tremors, most apparent in individuals that are above 50 years of age. The direct cause of this disease is still not fully understood, as it has been linked to decreased dopamine levels in some cases, and damage to the nervous system (dementia) in others.


The second disease I would like to research is Gaucher’s disease. What is interesting about this disease is that it could take either a neuronopathic form or a non-neuronopathic form. This disease is characterized by metabolic defects through an inherited deficiency of glucocerebrosidase due to mutations in the GBA1 (acid-β-glucosidase) gene. Out of all of the lysosomal storage disorders (LSDs), this disease remains the most prevalent worldwide.


The third disease I would like to look into is cystic fibrosis. This disease affects 70000 people worldwide, and leads to dangerous symptoms such as inflammation and tissue damage in the lungs. Research has linked this disease to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.



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The role of fatty acid β-oxidation in lymphangiogenesis

Acetyl-CoA has long been known for its importance as a metabolic intermediate for many pathways. For example, this intermediate can be used in anabolic pathways as in the case for fatty-acid synthesis in the cytosol of human cells, or could be a product of catabolic pathways like fatty-acid oxidation in the mitochondria. Most recently however, acetyl-CoA has been shown to be involved in other functions in the body, particularly with respect to the formation of lymphatic vessels and the process of lymphangiogenesis. In their recent publication in Nature, Wong et al. explain how lymphatic endothelial cells (LECs) use fatty-acid beta-oxidation (FAO) for differentiation and epigenetic regulation through histone acylation (Wong et al., 2016).

In order for FAO to occur, acetyl-CoA must first be transported from the vicinity of the cytosol across the mitochondrial membrane and into the mitochondrial matrix in the form of fatty-acyl CoA. This is done through the mechanism of the enzyme carnitine palmitoyl-transferase 1 (CPT1), which is the rate-limiting step for FAO. Wong et al. devoted their attention to this process in LECs, in which extracellular cues such as VEGF-C promote differentiation of these cells from venous endothelial cells (VECs). The importance of these cells lies in their contribution to normal physiology, as dysregulation can lead to many diseases that include cancer metastasis, organ transplant rejection, and lymphedema (Choi et al., 2012).

Wong et al. initiated their experiment by observing the expression levels of CPT1A in LECs vs. VECs, and found that these levels were higher in the former. This suggested that FAO was also higher in these cells, which led the authors to hypothesize that CPT1A may be directly required for lymphangiogenesis (Wong et al., 2016). This hypothesis was also supported when they performed CPT1A knockdown (CPT1AKD) in LECs, and observed reduced LEC proliferation and migration which led to lymphatic defects in vivo.

Beta-oxidation of fatty acids leads to the production of ATP; however, the authors noticed that CPT1AKD did not cause energy distress. Instead, fatty acids provided acetyl-CoA, which helped to sustain the Krebs cycle and deoxyribonucleotide (dNTP) synthesis for proliferation of LECs. As a result, the authors explored the possibility that acetyl-CoA generated by FAO in LECs might be involved in epigenetic modification of lymphangiogenic gene expression. They focused particularly on VEGFR3 transcription regulated by histone acetyltransferase (HAT) p300 through PROX1. Through their experiments, the authors observed that PROX1 does indeed bind p300 histone acetyltransferase to enhance H3K9 acetylation of LEC genes, which ultimately enables LEC differentiation. Furthermore, they observed that CPT1AKD reduced H3K9ac levels in pLECs to the levels seen in VECs, which supports that histone acylation is directly involved in the successful differentiation of LECs (Wong et al., 2016).

Finally, the authors also display a translational potential for their findings. They found that pharmacological blockade of CPT1 inhibits injury-induced lymphangiogenesis, and that replenishment with acetone actually rescues the defects due to FAO inhibition (Wong et al., 2016). As a result, these findings become of tremendous high impact considering the wide array of potential applications they can lead to, especially with respect to curing dangerous diseases and promoting lymphangiogenesis in pathological conditions such as lymphedema.

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My Choice in Biochemistry

Biochemistry is the study of vital chemical processes and applications in biological living systems. It allows us to understand the physiology of organisms at the biomolecular level, as it integrates the sciences of molecular biology, neuroscience, structural biology and biophysics to unravel the chemical basis of life in bacteria, plants and animals. As a child, I had always been fascinated by the complexity of chemistry and biology, more so than any other subject. Particularly, discussions in class regarding bacterial infections generated tremendous curiosity and remained as one of the most intriguing topics to me. This was partly due to my amazement in how they were so widespread in the local area where I lived in Syria, along with the dangers that these tiny microbes imposed to human beings all over the world. More closely, we had always learned that Gram-negative bacteria are much harder to kill than Gram-positive bacteria due to their possession of a unique outer membrane structure, which was somehow related to increased pathogenic behavior of these organisms. Because of this, I sought to strengthen my knowledge in understanding how microscopic structural differences between the two membranes impact the functionalities of these bacteria, and what chemical processes lead to those differences. Ultimately, this would segue into fulfilling my enthusiasm for biochemistry and attaining a job in the medical field as well.

Considering that conceptual learning was only partially satisfying to me, I determined to become more engaged in gaining hands-on experience in the biochemical and clinical fields. As a result, I first started off by shadowing a general practitioner in the area that I lived in. This experience was particularly helpful in allowing me to learn tremendous new medical terminology, observe the different symptoms that correspond to pathogenic infections (primarily bacterial) and get exposed to the names of the most common antibiotics used against these bacterial infections, including broad-spectrum penicillins and different cephalosporins. I even became more familiar with how these antibiotics may interfere with the bacteria and what structures they target, which made me even more eager to enhance my knowledge and pursue a biochemistry-rich, clinical career. Therefore, I also elected to become a volunteer in the intensive care unit of a local hospital a few years later. Here, I was enriched with tremendous knowledge with respect to enzymology and how medications enhance or interfere with biological systems at the protein level. The importance of enzymes in catalyzing an immense number of reactions in organisms become more obvious to me, which attracted me even further towards this field.

A couple years following my arrival to the United States, I joined Muhlenberg College as a pre-medical undergraduate student seeking to pursue a career in medicine. Aligning with my passion, I decided to major in biochemistry, and be further exposed to more difficult principles and concepts. Fortunately, the elite instructors and classes at Muhlenberg did not disappoint, but rather made me thankful for this decision. I progressively learned more about enzymes and their kinetics, and became more knowledgeable about incredible biochemical research and discoveries that relate to human health in particular. Amongst all of the great experiences at Muhlenberg, research under the supervision of Dr. Keri Colabroy was the most gratifying. Through developing new kinetic models for L-DOPA 2,3-dioxygenase from Streptomyces lincolnensis, an enzyme involved in the biosynthesis of the propylhygric acid moiety of the antibiotic lincomycin, I was part of potential future incentives in developing a lincomycin antibiotic derivative that is less resisted and more effective against certain bacterial strains than the current medication available. As a result, this directly tied with my ambitions to employ biochemical knowledge and techniques in combating infections and supporting my aspirations in becoming a doctor. Altogether, biochemistry is truly an astonishing field, and I am grateful to have developed a passion for a science that integrates and explains many chemical and biological aspects of life today, and helps me fulfill my dream in becoming a successful physician.


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