Bacterial Amyloid (Curli Fibers) Forms Biofilms

E. coli Curli Stacks in Congo Red Staining Fibers

We can’t cure diseases, because we don’t understand basic chemistry (what is hydrophobic) and biology (which came first the biofilm or the bacterial cell wall?)  Let’s look at a fundamental biological process, how bacteria form biofilms.

Biofilm Formation from Secreted Proteins and Polysaccharides

Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix.  The matrix stained red with Congo Red.

Congo Red Stains Amyloids, Cellulose and Rare LPS



Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red.  This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease.  But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide.  This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic.  That turns out to be a half-truth. 

Faces of Sugars Are Hydrophobic

The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds.  The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine.  Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues.  This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand.  Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.

Basic Amino Acids Bind Hydrophobically

Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic.  The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic.  Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate.  A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus.  Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin.  In each of these binding examples the binding is primarily hydrophobic.

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack.  In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber.  At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length.  The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red.  This same spacing of basic amino acids fits the sugars in heparin.  Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s

Bacterial Biofilms Form from Amyloids and Polysaccharides

The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers.  These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli.  It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots.  Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.

Do Biofilms Foment Amyloid Production?

Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces.  It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions.  The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production.  The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.

Berberine Binds Heparin and Blocks Amyloids and Biofilms

Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms.  There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.

Prostate, Prostatitis, Cancer, Causes and Cures

Prostate problems are pervasive, and progressive in the U.S. -- approximately a quarter of a million prostate cancers are diagnosed each year. Chronic inflammation due to age or diet enhances prostatitis and cancer.

Prostate Surrounds Male Urethra

The prostate is a sexual organ. It responds to sexual stimuli, both physical and hormonal, and as a consequence is vulnerable to the same hazards: infection, inflammation, cycles of elaboration and cancer. The prostate straddles the male urethra and inflammatory swelling of the prostrate can strangle the flow of urine from the bladder. The prostate contributes part of the seminal fluid and backward flow of bacteria from the urethra into the prostate can be a source of infection.

Prostate Cells Require Testosterone for Growth

The prostate tissue responds to testosterone. The ebb and flow of testosterone, associated with sexual activity, results in increased production of prostate fluid containing proteins and other components that enhance performance of sperm in the female reproductive tract. The bottom line here is that prostate tissue and derived prostate cancer, responds to testosterone. For this reason, prostate cancer treatment has been based on blocking testosterone stimulation by removal of sources of testosterone and blocking testosterone receptors.

Testosterone Starvation Is Prostate Cancer Treatment

Testosterone, estrogen and vitamin D receptors are all cytoplasmic and the hormone/receptor complexes act as transcription factors to alter nuclear gene expression. It would be expected that prostate cancer, as well as breast, cervical, ovarian, testicular, etc. will respond in a complex manner to steroid hormones, including androgens, estrogens and vitamin D.

Cancer Requires NF-kB Activation

The common biochemistry supporting all cancers is inflammation that is required for proliferation in tissue. Central to inflammation is the transcription factor NF-kB. Inhibitors that block the activation of NF-kB also stop cancer. Most of these blockers are effective against cancer cells grown in culture flasks, because the inhibitors are taken directly into the cells and make contact with NF-kB (or stabilize the NF-kB inhibitor IkB.) Curcumin from turmeric, is one of the most potent inhibitors of NF-kB activation. It is very effective in cultures, but is only modestly effective against inflammation when eaten.

It is difficult to treat secretory tissues, such as prostate, breast, uterus, etc., because much of the tissue is separated from the blood circulation. Thus, infections in these tissues are harder to treat with antibiotics.

Prostatitis Results from Urinary Infections

The prostate is prone to chronic infections. Thus, urinary tract infections (UTIs) can lead to prostate infections (prostatitis). These chronic infections can contribute to chronic systemic inflammation. One symptom of chronic inflammation is depression (treated with SSRIs, antidepressants) another symptom is premature ejaculation (also treated with SSRIs.) Prostatitis-based inflammation can also set the stage for cancer.

Transglutaminase Autoantigen

Autoimmune diseases are also associated with chronic inflammation. One of the common autoantigens in autoimmune disease is tissue transglutaminase (tTG). Celiac is a classic example of the involvement of tTG, since tTG acts on the glutamine amino acids of gluten and converts them into glutamic acid residues. In the process tTG becomes covalently attached to the gluten. The strong heparin binding domains of tTG also facilitate its uptake and processing as an immunogen under inflammatory conditions and result in antibody production to both tTG and gluten. Anti-tTG antibodies and inflammation can also lead to attack on other tissues, such as the thyroid and skin, leading to a variety of celiac-associated autoimmune conditions.

The prostate produces its own transglutaminase. I think it is likely that prostatitis in some cases progresses to an autoimmune disease and prostate transglutaminase is a likely candidate for one of the autoantigens involved. This also predicts an association with celiac and a requirement for chronic systemic infection with a likely elevation of C-reactive protein and inflammatory cytokines (IL-1, IL-6, TNF). Deficiency of vitamin D and omega-3 fatty acids are probably major contributors. Increased risk attributed to consumption of a high fat diet and meat, is probably actually due to inflammation from a high carbohydrate diet and high omega-6 vegetable oils (or perhaps corn-fed meat.)

Protect Prostate with Anti-Inflammatory Diet

Prostate problems are frequently assumed to be a natural result of advancing age, but they are actually symptoms of mismanaged chronic inflammation. Men should not just stand and wait for prostate problems.

Avoidance and treatment of prostate problems seems to be an obvious application for an anti-inflammatory diet and lifestyle.

Extreme Flu Remedies

Experimental Therapies for ARDS, Cytokine Storms

Do not do this at home. There are doctors and hospitals. Use them.

....But, if a doctor emailed me pleading for any ideas that I had to save a bunch of patients suffering from acute respiratory distress syndrome (ARDS) from Tamiflu-resistant H1N1, my first response would be to suggest therapies designed for ARDS from other origins, e.g. burns, septicemia, etc.

Cytokine Storms Are Out of Control
When too much tissue is injured, the local, molecular communication that normally occurs just between cells, spills into the blood stream and becomes potentially lethal. That is what happens in anaphylactic shock. It is also what happens in cytokine storms, where inflammatory cytokines that are normally short-lived and processed locally to progress into recovery, erupt into the blood stream and impact distant organs.

Major disruption of body function by aggressive blood infections or burns over most of the body, will be lethal without heroic medical interventions. These are injuries beyond the evolved adaptations of mammals.  Until recently there were no survivors.

Influenza has been around for a long time. Humans, other mammals and birds get the flu and get over it. Many body cells become infected, antibodies specific to the virus are produced within about a week, the infected cells are killed, the virus is digested and life goes on.

People die from the flu, because an opportunistic pathogen causes a lethal secondary infection, or the body over-reacts and damages itself in attempts to attack its own infected cells. This is a cytokine storm.

Silence the Storms
Cytokine storms can be weathered by blocking the signaling system. Cytokines are just small proteins that are complementary in shape to corresponding protein receptors that penetrate through the surface membranes of cells throughout the body. Binding of cytokine to receptor changes the shape of the receptor and transmits a signal into the cytoplasm of the receptive cell. This turns on aggressive behavior of immune cells and triggers more inflammatory signaling in other cells. This causes fever, malaise, etc.

...But, I was the one the doctor is pleading with to save the people. And I know that there is more to cytokine signaling than just cytokines and receptors. There are also heparan sulfate proteoglycans (HSPGs). Cytokines are not supposed to be broadcast throughout the body. Cytokines function in the space between cells, the extracellular matrix. Polysaccharides attached to membrane proteins, HSPGs, are secreted at one end of the cells, sweep across the surface and are taken back up at the other end. Cytokines have heparan-binding domains and so they stick to the heparan and are swept along. Cytokines can move from one cell to another as the sweeping HSPGs of adjacent cells come in contact.

HSPGs Mediate Cytokine Signaling
The critical point here is that cytokines bind to their receptors with the heparan between -- the cytokine and receptor are like two halves of a bun and the hot dog is the heparan. In fact the heparan bridges two cytokine/receptor complexes to make an active, signaling pentamer.

Heparin Can Block Cytokine Signaling
Heparin is a fragment of heparan sulfate produced by enzymatic degradation of HSPG. Commercial heparin, used to block blood clotting, is obtained from the mast cells of lungs and intestines of hogs and cattle. The mast cells release heparin and histamine in response to parasites or pollen. Since heparin is a short version of heparan sulfate, it can block the formation of active cytokine/receptor complexes.

Heparin is used in a mist to treat the lungs of burn patients. It is also injected into some infertility patients to suppress inflammation that is inhibiting implantation and gestation. It is also effective in treatment of autoimmune inflammation in Crohn’s disease. I think it should be tested as a therapy for H1N1 cytokine storms. It may be useful in nebulizing mists and oral treatment of intestines.

Berberine Binds to HSPG
Berberine is a phytochemical from Barberry traditionally used in the treatment of intestinal infections and arthritis. It also binds to heparan sulfate to form fluorescent complexes visible in microscopy. Berberine-treated mast cells glow brightly. Heparan sulfate can also be detected in Alzheimer’s plaque, atherosclerotic plaque and prion complexes. Because berberine binds to heparan sulfate, it should also disrupt cytokine signaling. It has been used successfully in treatment of septicemic ARDS.

Curcumin Blocks NFkB
One of the most potent chemicals that blocks inflammatory signaling via the inflammatory transcription factor, NFkB, is curcumin. Curcumin is a major component of the spice turmeric. Oral curcumin can be enhanced by co-administration of black pepper, because the piperine in pepper inhibits intestinal inactivation.

Anti-Inflammatory Diet
Of course, I would also recommend vigorous implementation of an anti-inflammatory diet and lifestyle to support any medical treatment.

Allergy, Asthma, Autoimmunity Start the Same Way

Inflammation is the current medical buzzword. Name the disease and inflammation is there.

Reproduction Requires Controlled Inflammation
Aspirin blocks many of the steps in triggering inflammation and thus, aspirin administration can be used to reveal a role of inflammation in many unexpected places. Aspirin is effective in blocking some forms of infertility, inhibiting miscarriages and ameliorating postpartum depression. So inflammation is a critical part of reproduction. But, also notice that depression is a symptom of chronic inflammation.

Cancer Requires Inflammation
High dose (IV) aspirin has been successfully used to treat cancer. Inflammation is required for cancer growth, because both use the same transcription factor, NFkB. The aberrant signaling of cancer cells would normally lead to programed cell death, apoptosis, but inflammation blocks apoptosis. Aspirin can in turn block NFkB and in the absence of inflammation, cancer cells die by apoptosis.

Inflammation is Self-Limiting
Aspirin also transforms the COX/lipoxidase system to produce anti-inflammatory prostaglandins/eicosinoids. Inflammation normally progresses into anti-inflammation. Blocking this progression leads to chronic inflammation and a shift from local to systemic inflammation with the rise of inflammatory interleukins in the blood stream.

Immune Response Requires Inflammation
The signal molecules (IL-1, IL-6, TNF) and transcription factor, NFkB, associated with inflammation were all initially identified in the development of lymphocytes. Hence, IL stands for interleukin, a hormone that triggers leukocyte (literally white blood cells or cells associated with the lymphatic immune system, i.e. lymphocytes) development. The nuclear factor, i.e. transcription factor, involved in expression of the large chain, kappa, of immunoglobulins in B cells, was called NFkB.

Genes Expressed by NFkB Cause Symptoms of Inflammation
About five dozen genes are under control of NFkB. Among these are COX-2, the enzyme that converts omega-6 arachidonic acid to inflammatory prostaglandins; iNOS, the enzyme that produces nitric oxide that dilates blood vessels to produce hot, red skin; and the inflammatory interleukins, IL-1, IL-6 and TNF, associated with autoimmune disease, fatigue and cachexia (wasting).

Autoimmunity and Allergy Start with Inflammation
Medical treatments focus on symptom abatement and ignore cause. What causes obesity, allergy or autoimmune disease? The answer appears to be chronic systemic inflammation plus exposure to unusual proteins. The unusual proteins are immunogenic, i.e. interact with the immune system to produce antibodies or reactive T-cell receptors, and are subsequently recognized as autoantigens or allergens, that are the targets for immune attack. Inspection of these autoantigens and allergens shows that they all have one thing in common, they bind to heparin via a strong heparin-binding protein domain that is typically a triplet of adjacent basic amino acids.

Heparin is a Short, Highly Sulfated Fragment of Heparan Sulfate
Commercial heparin is purified from the intestines of hogs and cattle. Heparin is released from mast cells (made fluorescent for microscopy using berberine) along with histamine and is released into the intestines to block pathogens from binding to the heparan sulfate that is part of the intestine surface. The heparin is anti-inflammatory and it contributes to minimizing the inflammatory response of the intestines to food.

Inflammation Reduces Heparan Sulfate Production
Pathogen-generated inflammation of the intestines reduces heparan sulfate production and increases immune response to food antigens. NFkB activation by inflammation turns off the production of some genes needed for heparan sulfate proteoglycan (HSPG) synthesis. Since HSPG is a major component of the basement membrane that holds tissues together, the reduction of HSPG results in protein loss (proteinuria) from kidneys, leaking of intestines, and disruption of the blood/brain barrier.

Reduction of HSPG Results in Immunological Presentation of Autoantigens/Allergens
Proteins are brought into cells by specific binding to protein receptors. In many cases, particularly involving signaling or growth factors, both the signal molecules and the receptors bind to heparin. In addition, there is a robust circulation of HSPG, which is secreted and internalized with a half-life of approximately six hours. The sweep of the HSPGs take heparin-binding proteins with them for internalization, e.g. HIV-TAT, heparanase, tissue transglutaminase. I think that this HSPG sweep under inflammatory conditions also internalizes basic autoantigens and allergens with strong heparin-binding domains. This internalization is the first step toward immunological presentation and the immune response to autoantigens and allergens.

Autoantigen/autoantibody/HSPG Complexes Kill Cells
Antibodies against self-antigens, autoantigens form antigen/antibody complexes that also bind to and cross-link HSPGs, because of the heparin-binding domains of the autoantigens. The large complexes may disrupt HSPG circulation and trigger apoptosis or abnormal physiology. There are many other examples of heparin-based complexes that are toxic, e.g. Alzheimer’s amyloid plaque, diabetic beta cell antibody complexes, celiac gluten/tRG antibody complexes, multiple sclerosis myelin antibody complexes, atherosclerotic plaque.

Anti-Inflammatory Diet and Lifestyle Protects
Dietary and lifestyle adjustments that minimize inflammation, e.g. low starch, no HFCS, low vegetable oil (except olive) and supplements of vitamins D & C, fish oil (omega-3) and glucosamine, reduce the risk of allergies/asthma, degenerative diseases and cancers. Simple, high level supplements with fish oil reduce numerous mental disorders, e.g. depression, ADHD; infertility, pre-eclampsia and postpartum depression; allergies, asthma; arthritis, atherosclerosis; burn recovery, septicemia and head injury.

Reducing Inflammation is a Panacea for Modern Diseases
Most modern diseases have an inflammatory component, because modern diets are rich in inflammatory components, e.g. starch/sugar, corn/soy oil, HFCS, trans fats, and exercise is minimal. The medical industry has not successfully promoted healthy eating and exercise; and in fact has promoted the devastating replacement of saturated fats with inflammatory polyunsaturated vegetable oils. Meat production has moved away from grazing on omega-3-rich plant vegetation to omega-6-rich corn and soy. Replacement of the corn/soy based agricultural economy would have predictably immense beneficial impact in reducing inflammation-based degenerative autoimmune diseases and cancers.

Synuclein and Amyloid Diseases

NSAIDs, such as ibuprofen and aspirin are possible treatments to inhibit the aggregation of proteins (synuclein, beta amyloid) on charged polymers in amyloid diseases, such as Parkinson’s disease, Alzheimer’s disease, etc. Contradictory studies show that intracellular aggregate formation may be protective, since dimers are more toxic than aggregates.

The list of amyloid diseases is long and there are few effective treatments. In each case a protein starts to accumulate in fibers that form amyloid plaques inside or outside the cells. The large aggregates outside are toxic. Inside it appears that the large aggregates are not as toxic as small clumps, oligomers, of the protein.

The amyloid proteins are stacked up in the fibers in a very organized way, so that the same portions of the protein are lined up on each side of the fibers. Outside the cell, the regions with basic amino acids interact with heparin, and in Alzheimer’s disease, for example, the beta amyloid plaque is half heparin. In test tube experiments, fiber formation from protein solutions is accelerated by adding heparin.

Amyloid fibers also form inside cells in the case of the tau fibers of Alzheimer’s disease or the synuclein aggregates in Parkinson’s disease. In theses cases, there should not be any intracellular heparin, and it is not known what polyanion (RNA?) serves to accelerate fiber formation in these cases.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin, reduce the incidence of Parkinson’s and Alzheimer’s diseases. It has recently been shown that in test tube experiments, NSAIDs also decrease the formation of amyloid fibers from synuclein.

Amyloid fibers can be stained by Congo Red and thioflavin. Curcumin is the active component of tumeric and it has a structure related to Congo Red. Curcumin has been shown in recent studies to block synuclein amyloid formation.

In addition, the heparin in the fiber complexes can be stained with berberine. Berberine is a traditional herbal treatment for arthritis. It would not be surprising if it was also effective against Alzheimer’s amyloid plaque.

The large extracellular plaque aggregates appear to be toxic, but the small, oligomeric aggregate of protein appear to be the toxic form in cells. Recent experiments show that facilitating the formation of large intracellular aggregates minimizes the toxicity in animal models of Huntington’s and Parkinson’s diseases. It appears that the large visible aggregates are not the form that kills the cell.

For the time being, the only safe treatments that focus on amyloid fiber formation are the NSAIDs, curcumin and perhaps berberine.

references:
Hirohata M, Ono K, Morinaga A, Yamada M. 2008. Non-steroidal anti-inflammatory drugs have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro. Neuropharmacology 54(3):620-7.

Pandey N, Strider J, Nolan WC, Yan SX, Galvin JE. 2008. Curcumin inhibits aggregation of alpha-synuclein. Acta Neuropathol. 115(4):479-89.

Bodner RA, Outeiro TF, Altmann S, Maxwell MM, Cho SH, Hyman BT, McLean PJ, Young AB, Housman DE, Kazantsev AG. 2006. Pharmacological promotion of inclusion formation: a therapeutic approach for Huntington's and Parkinson's diseases. Proc Natl Acad Sci U S A. 103(11):4246-51.

Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet JC, McLean PJ, Young AB, Abagyan R, Feany MB, Hyman BT, Kazantsev AG. 2007. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 317(5837):516-9.

Topoisomerase Inhibitors

Inhibiting enzymes involved in DNA synthesis should stop cancer cells, because cancer is uncontrolled cell division. Topoisomerases are enzymes that help to relieve the twists on double helical DNA as it unwinds preparatory to replication. It appears logical that topoisomerase inhibitors should be cancer inhibitors. Unfortunately targeting DNA-binding proteins also targets most of the signal receptors that are the targets for the evolution of plant alkaloids.

Drugs are designed to be specific in their interactions with a particular target protein, but they are too small to be specific and end up binding to many other related proteins. Hence, drugs have side reactions that are to some extent unpredictable, because the interacting proteins are not known.

Aspirin, for example, is supposed to bind to and inhibit COX-2, the enzyme that converts omega-3 and omega-6, long-chain fatty acids into corresponding anti-inflammatory and inflammatory prostaglandins, resp. Aspirin also binds to proteins that inhibit NFkB, the transcription factor that controls expression of inflammatory genes. Aspirin binds to dozens of other proteins. Aspirin does lots of other things than just blunt inflammation, but those side reactions are usually not significant enough to get our attention.

Heparin is one of the most commonly used drugs. It binds to and activates an inhibitor of thrombin, an enzyme that activates fibrin and mediates clotting. Heparin also binds to other components of the clotting system, as well as a dozen components of the complement system, and most of the cytokines that control communications throughout the body. When patients are given heparin injections, heparin binds continually to all of these components and must be constantly supplemented and monitored. Inflammation depletes the heparin components throughout the body, so it is not known prior to injection, how much heparin will be needed to saturate other serum proteins before the desired level of clotting inhibition is achieved. This illustrates rather dramatically that most drugs have only limited specificity.

One of my students provided another example of the minimal specificity of small molecules, especially the alkaloids and phenolics produced by plants. He brought to me a research article espousing the use of phenolics from yerba mate, which serves as a coffee-like stimulant in Argentina, as a topoisomerase inhibitor and potential anti-tumor treatment. Sure enough, phenolics extracted from this plant inhibit topoisomerase, and they may well be able to inhibit the growth of tumors, but it is doubtful that the binding of the phenolics to topoisomerase in the tumor nuclei has anything to do with inhibition of tumor growth.

Topoisomerase binds to nuclear DNA as the DNA unwinds during replication to produce two new double helical DNA molecules. Topoisomerase is a DNA-binding protein, i.e. a protein that binds to a negatively charged polymer of small deoxyribose sugars and flat purine and pyrimidine bases. Proteins bind to DNA in two ways. Amino acids of the protein either bind along the edges of the hydrophobic stack of base pairs, e.g. sequence-specific transcription factors, or they provide hydrophobic, flat surfaces that bind to the hydrophobic faces of the separated bases. Topoisomerase does both, because it deals with single-stranded regions of DNA and therefore binds to both the phosphates, as well as the bases. The important point here is that both aromatic amino acids, with flat hydrophobic rings, and the hydrophobic tails of basic amino acids, i.e. lysine and arginine, bind to the hydrophobic faces of nucleic acid bases.

I have illustrated the binding of a “topoisomerase inhibitor” to show the arginine (blue) in the active site cleft of the topoisomerase that binds across the hydrophobic face of the inhibitor (grey and red). Many plant phenolics and alkaloids would be expected to similarly bind and act as inhibitors of topoisomerase. This observation and the ease by which alkaloids enter cells (attached to circulating heparan sulfate?) suggests that a major function of the nuclear envelope may be to minimize access of alkaloid and related molecules to the nucleic acid binding proteins of the nucleus.

The binding promiscuity of secondary plant products is further exemplified by berberine. Berberine is an alkaloid found in goldenseal and is an herbal remedy used to treat a variety of inflammatory diseases. It also binds to heparin (and nucleic acids) to produce a fluorescent complex. Thus, mast cells that store and secrete histamine and heparin to produce the symptoms of allergy, can be vividly stained with berberine.

I could not resist the temptation to check to see if berberine also binds to topoisomerase. A quick search of the research literature showed that berberine is in fact a topoisomerase inhibitor.

The numerous cross reactions of drugs are further illustrated by metformin, the common drug used in the treatment of type II diabetes. Metformin is approximately planar and provides a surface that cannot hydrogen bond, i.e. it is hydrophobic. I expected that metformin would bind to tryptophans that I observed as common substrate-binding amino acids in the active sites of proteins that bound to polysaccharides, e.g. lectins, glycosidases and glycanases. To test this, I had students in one of my courses examine the inhibitory activity of metformin on E. coli beta-galactosidase. They found measurable inhibition and support for competitive binding to the active site that contains a pair of the predicted tryptophans.

My protein modeling and structural studies show the basis for numerous interactions between plant secondary compounds, drugs, nucleic acids, polysaccharides (glycosaminoglycans, e.g. heparin) and proteins. Unpredicted cross reactions abound and every drug can be expected to interact with multiple proteins. This provides a note of caution to the use of any drug and encourages minimal exposure, since many unobserved and unanticipated side effects are occurring. These observations also question routine ingestion of herbal remedies, after all, plants use their secondary products as potent defenses against being eaten. Alkaloids disrupt nervous systems and cellular signaling. Plants are not naturally safe.

Mast Cell Heparin

Mast cells are sentinels in tissues. They respond to invading pathogens by releasing their stored histamine, enzymes and heparin. The heparin modifies the activity of enzymes and cytokines.

What are mast cells and why are they loaded with heparin (left)? Mast cells start in the bone marrow, like many other components of the immune system. They then move into the blood stream and offload in most of the tissues that typically encounter pathogens and parasites. Thus, the typical commercial source of the mast cell-produced heparin is pig intestines or cow lungs, i.e. since heparin is made and stored in mast cells and mast cells are abundant in lungs and intestines, those are the sources of crude heparin. Proteins bound to the crude heparin are removed as the heparin is cleaned up to be used as an anti-clotting drug.

Mast cells are sentinels near the surface of mucus membranes that line the airways of the lungs and the digestive tract. Diseases of the lungs and intestines, e.g. asthma and inflammatory bowel disease, that have an inflammatory and/or autoimmune component yield high levels of mast cells in the affected tissues. Pathogens or parasites coming in contact with mast cells trigger the sudden release of vesicles full of histamine, enzymes and heparin.

Heparin stored in vesicles in mast cells can also be readily visualized by staining the mast cells in microscope sections using the fluorescent dye berberine (left). Berberine binds quite specifically to heparin and is also used in herbal medicine as a treatment for many inflammatory diseases, such as arthritis. It would be very interesting to know whether berberine has any effect on asthma.

Mast cells display a variety of receptor proteins on their surfaces. Protein receptors work by binding target molecules, ligands, changing their shapes and transmitting a signal through the cytoplasm. A key aspect of the signal transmission is the requirement for the ligand binding to bring together receptors in pairs. The pairing of receptors during ligand binding is facilitated by the binding of heparin to both ligands and receptors. Two ligands, e.g. cytokine peptides, such as TNF, can bind to adjacent sites on a heparin molecule and this pair can then bind to two receptors brought together on the surface of a cell. The receptors bind to the ligand and to the heparin. Some ligands will bind to their receptors without heparin, but the presence of heparin greatly accelerates and intensifies the reactions.

Heparin is synthesized in the vesicles of mast cells and binds to enzymes, e.g. tryptase, also present in the vesicles. The tryptase enzyme proteins form tetramers with heparin wrapped around the edge (left, edge view showing one pair of tryptase proteins with heparin bound diagonally to blue heparin-binding domains; other pair of tryptase proteins is hidden).

Interestingly the active site for each tryptase in the tetramer faces a hole where the four proteins come together. Thus the tetramer can degrade small peptides, but large proteins cannot get access to the blocked active sites. Monomers change shape and are no longer active.

Activated mast cells release their vesicle contents with some enzymes active and their bound heparin is replaced by the heparan sulfate attached to adjacent cells. Other enzymes are initially inactive bound to heparin and are activated by dissociation of the heparin once they are released from the vesicles. In both cases some of the heparin is released from the mast cells into the surrounding tissue. The free heparin can bind to cytokines released from other cells and the combined pairs of cytokines bound to heparin can in turn bind to appropriate receptors on other cells. The abundance of heparan sulfate bound to other cells will determine whether additional heparin is required for receptor responses from particular cytokines. Cells with abundant heparan sulfates will sweep heparin binding ligands toward receptors aggregated in lipid rafts, as the heparan sulfate proteoglycans are internalized for recycling.

Mast cells can be activated by allergens, because of IgE receptors. IgEs are antibodies that trigger allergic responses. The IgEs produced by antibody producing B lymphocytes circulate in the blood serum and bind to mast cell receptor proteins. Allergen molecules bind to the IgE-receptor complexes, trigger the activation of the mast cells and release histamine. The histamine binds to receptors on other cells and produces the symptoms of allergy or asthma.
Heparin can be sprayed into the lungs of asthma sufferers and reduce symptoms. This suggests that the ratio of heparin to cytokines is important and that cytokine signaling required for asthma episodes of airway constriction can bind individually to different heparin molecules and minimize mast cell triggering and histamine release.

Asthma also responds to a general decrease in chronic systemic inflammation. Thus, an anti-inflammatory diet and lifestyle, can reduce episodes and potentially reverse symptoms. Omega-3 oils and glucosamine, for example are both effective.

Tryptase model: Sommerhoff CP, Bode W, Pereira PJ, Stubbs MT, Stürzebecher J, Piechottka GP, Matschiner G, Bergner A. 1999. The structure of the human betaII-tryptase tetramer: fo(u)r better or worse. Proc Natl Acad Sci U S A 96(20):10984-91.


Berberine staining of mast cell heparin: Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, Takahashi HK, Morgan ES, Dvorak AM, Fehling HJ, Rodewald HR. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 25:6199-210.