Blog

Reverse Transcriptase and Coronaviruses

See the source image

Picture by Wikimedia Commons

Articles review

This abstract comprises a simple analysis and description of the virus genome.

Human coronaviruses, or HCoVs consist of HCoV-229E and HCoV-NL63 in the Alphacoronavirus family and HCoV-OC43 and HCoV-HKU1 in the Betacoronavirus family.

Four CoVs, for instance, HKU1, NL63, 229E and OC43, are mainly responsible for mild respiratory disorders in human circulation.

SARS-CoV-2 is a β-coronavirus carrying a single positive RNA strand as genetic material, it has a lipidic envelope that confers an elliptic morphology.

CoVs evolved a relatively complex multiplication mechanism to facilitate virus reproduction. In their viral life cycle CoVs transmit genomes and subgenomic RNAs only from RNA templates, and do not need a step of DNA. CoVs use exonuclease NSP14, the first known RNA virus-encoded proofreading enzyme that in comparison with other error-prone RNA viruses, could be adapted to handle CoVs‘ large RNA genome.

Source: Genetic analysis of the 2019 coronavirus pandemic with from real-time reverse transcriptase polymerase chain reaction (nih.gov)

From original article report:

“Prolonged SARS-CoV-2 RNA shedding, and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious”. The researchers in this article have investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences may be responsible for PCR-positive tests.

In support of this hypothesis, they found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome.

To experimentally corroborate the possibility of viral retro-integration, they describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase, RT from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed.

-This is, of course, referred to an infection with SARS-CoV-2, and not to the genetic material (mRNA) introduced with the vaccine-

Source:

SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome | bioRxiv

In this last article slightly modified and reduced from myself also is commented about the reverse transcription of SARS-CoV-2.

The authors report that: “The researchers explored the occurrence of reverse transcription of the SARS-CoV-2 RNA into the human genome. This would result in positive PCR tests due to the continuing transcription of viral RNAs.

Reverse transcriptase activity has been detected within human cells, so as integration of the reverse transcription products.

The endogenous RT is potentially present in the form of human LINE-1 elements, which make up 17% of the human genome. These are autonomous retrotransposon elements that can transpose themselves as well as other elements of the genome back into the DNA of the nucleus for future transcription.

The researchers looked at the published RNA-sequences from SARS-CoV-2 infected cells, their purpose was to find chimeric transcripts, fusing human and viral RNA into the same genome. They found a good number of these in several different cell types, and from cells recovered from the bronchoalveolar lavage fluid obtained from COVID-19 patients.

The proportion of these chimeric sequences was directly correlated with the level of viral RNA in each sample. The greatest proportion was in cells recovered from the bronchoalveolar lavage fluid of severe COVID-19 patients, while there were almost none in blood cells.

Most of the host-viral chimeric protein contained the nucleocapsid sequences, as expected since this is the most abundant viral particles. This would, therefore, be the most likely to be reverse transcribed and then integrated. These findings for the researchers support the occurrence of this event within infected cells.

They conducted then an experiment inducing the overexpression of human LINE-1 elements or HIV-1 RT, in the cell line.

These cells were then infected with SARS-CoV-2. At two days post-infection, they carried out polymerase chain reaction, PCR, tests to detect the viral sequences, using the N-targeting primer sets used in the commonly used COVID-19 PCR tests.

PCR amplification of the purified cell DNA from infected cells showed the presence of the N protein bands. This did not occur in non-transfected or uninfected cells.

They also conducted an in vitro RT experiment, which showed that cell lysates from cells expressing RT of either type could cause reverse transcription of purified viral RNA from infected cells.

Using fluorescent in situ hybridization, FISH, technology, they trapped down the presence and ongoing transcription of the viral N sequences within the cell nucleus with the help of Ntargeting fluorescent probes. The N sequences were found in the cytoplasm, as expected of cells infected by SARS-CoV-2.

However, FISH also picked up N RNA signals from the nucleus of cells that overexpressed LINE-1, showing that integrated N sequences in the host genome were being transcribed there.

The integrated sequences are probably sub-genomic and cannot produce live infectious virions. This explains the positivity of later PCR tests for viral RNA in clinically recovered patients.

The authors suggest that the site of insertion and regulation by epigenetic factors, besides the existing immune state of the patient, may affect the translation of these sequences and their possible clinical consequences.

In conclusion, the study suggests that many PCR positive results could be due to viral transcripts from chimeric sequences instead of reflecting the presence of replicating virus in the host. If validated, this will require better tests to be used when assessing the efficacy of COVID-19 therapies in clinical trials, for example, in the future.

Source:

SARS-CoV-2 RNA can be reverse-transcribed (news-medical.net)

-As we can see, the role of the immune system and of the epigenetics factors is one of the major compounds in any disease, and especially considered with Covid-19 infections-

Thanks for Reading

Mariarosaria M.

History, Classification and Origins of Coronaviruses

See the source image

History

It looks like that the earliest reports of a coronavirus infection in animals took place in the late 1920s, when an acute respiratory infection of domesticated chickens emerged in North America. The virus responsible that caused the infection was isolated in 1933 and was then known as infectious bronchitis virus, IBV. New researchers cultivated the virus for the first time in 1937 and the specimen was known as the Beaudette strain. In the late 1940s, two more animal coronaviruses, JHM that causes brain disease in mice, and mouse hepatitis virus, MHV were discovered. At the time it was still not evidence that these three different viruses were related.

Human coronaviruses were discovered in the 1960s using two different methods in the United Kingdom and the United States. Scientists working at the Common Cold Unit of the British Medical Council Research collected a unique common cold virus denominated B814 in 1961. The virus could not be cultivated using standard techniques as those used for rhinoviruses, adenoviruses, and other known common cold viruses.

In 1965, the British scientists successfully cultivated the novel virus by serially passing it through organ culture of human embryonic trachea. The isolated virus when inoculated intranasally into volunteers caused a cold and was inactivated by ether which pointed to the presence of a lipid envelope. Scientists at the University of Chicago isolated a novel cold from medical students in 1962, they isolated and grew the virus in kidney tissue culture, denominating it 229E. The novel virus caused a cold in volunteers and, like B814, was inactivated by ether.

Scientists at St. Thomas Hospital in London, collaborating with those from the Medical Council Research in 1967 compared the structures of IBV, B814 and 229E using electron microscopy. The three viruses were shown to be morphologically related by their general shape and distinctive club-like spikes

A research group at the National Institute of Health the same year was able to isolate another member of this new group of viruses using organ culture and entitled one of the samples OC43, OC is for organ culture. Like B814, 229E, and IBV, the novel cold virus OC43 had distinctive club-like spikes when observed with the electron microscope.

The IBV-like novel cold viruses were soon shown to be also morphologically related to the mouse hepatitis virus. This new group of viruses were named coronaviruses after their distinctive morphological appearance. Other human coronaviruses have since been identified, including SARS-Covid in 2003, HCoV-NL63 in 2003, HCoV-HKU-1 in 2004, MERS-Co-V in 2013, and SARS-CoV-2 in 2019. There have also been many animal coronaviruses identified since the 1960s.

Classification

Coronaviruses belongs to the family of Coronaviridae, subfamily Orthocoronavirinae, order Nidovirales, and realm Riboviria. They are divided into the four genera: Alpha-coronavirusBeta-coronavirusGamma-coronavirus, and Delta-coronavirus. Alfa-coronaviruses and Beta-coronaviruses infect mammals, while Gamma-coronaviruses and Delta-coronaviruses primarily infect birds.

The genus alfa-coronavirus includes the Human coronavirus 229E and Human coronavirus NL63 among other animal coronaviruses

To the beta-coronavirus belong, the Human coronavirus HKU1, and OC43, MERS, SARS-CoV, and SARS-CoV-2 among other animal coronaviruses.

Delta and gamma genus includes only animal coronaviruses, (As of Wikipedia literature).

CDC Classification of Coronaviruses types

Alpha-coronavirus– 229E, Alpha-coronavirus– NL63

Beta-coronavirus– OC43, Beta-coronavirus– HKUI

-These are not to related to the variants of SARS-CoV-2, but to the genera of the coronaviruses family, variants are due to mutations of the same virus and are designed with same Greek alphabetical letters-

Origin

The most recent common ancestor of all coronaviruses is estimated to have existed as 8000 BCE, some models place the common ancestor even as far back as 55 million years or more, suggesting long term coevolution with bat and avian species. Bats and birds, as warm-blooded flying vertebrates, are an ideal natural reservoir for the coronavirus gene pool.

It seems that bats were the reservoir for alpha-and beta-coronavirus, and birds the reservoir for gamma and delta-coronaviruses. The large number and global range of bat and avian species that host viruses have facilitated massive evolution and dissemination of coronaviruses.

Many human coronaviruses have their origin in bats. MERS-CoV emerged in humans from bats through the intermediate host of camels. MERS-Cov, although related to several bat coronavirus species, appears to have diverged from these several centuries ago. The most closely related bat coronavirus and SARS-CoV diverged in 1986. The ancestors of SARS-CoV first infected leaf-nose bats of the genus Hipposideridae; subsequently, they spread to horseshoe bats in the species Rhinolophidae, then to Asian Palm civets, and finally to humans.

Unlike other beta-coronaviruses, bovine coronavirus of the species Beta-coronavirus-1 is thought to have originated in rodents and not in bats. In the 1790s, equine coronavirus diverged from the bovine coronavirus after a cross-species jump. Later in the 1890s, human coronavirus OC43 diverged from bovine coronavirus after another cross-species spillover event. It is speculated that the flu pandemic of 1980 may have been caused by this spillover event, and not by the influenza virus, because of the related timing, neurological symptoms, and unknown causative agent of the pandemic.

Besides causing respiratory infections, human coronavirus OC43 is also suspected of playing a role in neurological diseases. In the 1950s, the human coronavirus OC43 began to diverge into its present genotypes. Phylogenetically, mouse hepatitis virus, murine coronavirus, which infects the mouse’s liver and central nervous system is related to human coronavirus OC43 and bovine coronavirus. Human coronavirus HKU1, like the above-mentioned viruses, also has its origins in rodents.

Infection in Humans

Coronaviruses vary significantly in risk factor, they can cause colds with major symptoms, such as fever, and a sore throat from swollen adenoids, they can cause pneumonia, either direct viral pneumonia or secondary bacterial pneumonia, and bronchitis, either direct viral bronchitis or secondary bacterial bronchitis. The human coronavirus discovered in 2003, SARSCoV, which causes severe acute respiratory syndrome, SARS, has a unique pathogenesis because it causes both upper and lower respiratory tract infection.

Six species of human coronaviruses are known, with one species subdivided into two different strains, making seven strains of human coronaviruses in total.

Four human coronaviruses produce symptoms that are generally mild, even though it is argued they might have been more aggressive in the past:

  1. Human coronavirus (HCoV-OC43), β-CoV
  2. Human coronavirus HKU1 (HCoV-HKU1), β-CoV
  3. Human coronavirus 229E (HCoV-229E), α-CoV
  4. Human coronavirus NL63 (HCoV-NL63), α-CoV

Three human coronaviruses produce potentially severe symptoms:

  1. Severe acute respiratory syndrome (SARS-CoV), β-CoV (identified in 2003)
  2. Middle East respiratory syndrome related coronavirus (MERS-CoV), β-CoV (identified in 2012)
  3. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), β-CoV (identified in 2019)

These cause the diseases commonly known as SARS, MERS, and COVID-19 respectively.

Common cold

Although the common cold is usually caused by rhinoviruses in about 15% of cases the cause is a coronavirus. The human coronaviruses HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63 continually circulate in the human population in adults and children worldwide and produce the generally mild symptoms of the common cold. The four mild coronaviruses have a seasonal incidence occurring in the winter months in temperate climates. There is no preponderance in any season in tropical climates.

Severe Acute Respiratory Syndrome, SARS

In 2003, following the outbreak of severe acute respiratory syndrome, SARS was developing in Asia, and secondary cases elsewhere in the world, the World Health Organization, WHO, released a statement where was tested that a novel coronavirus identified by several laboratories was the causative agent for SARS. The virus was officially named the SARS coronavirus, SARS-CoV.

Middle East Respiratory Syndrome, MERS

In September 2012, a new type of coronavirus was identified and officially called Middle East respiratory syndrome coronavirus, MERS-CoV. The WHO issued a global alert soon after, and update in September 2012 that the virus did not seem to pass easily from person to person. However, in May 2013, a case of human-to-human transmission in France was confirmed by the French Ministry of Social Affairs and Health. In addition, cases of human-to-human transmission were reported by the Ministry of Health in Tunisia. Despite this, it appears the virus had trouble spreading from human to human, as most individuals who are infected do not transmit the virus. 

After the Dutch Erasmus Medical Center sequenced the virus, the virus was given a new name, Human Coronavirus—Erasmus Medical Centre, HCoV-EMC. The final name for the virus is Middle East respiratory syndrome coronavirus, MERS-CoV. The only U.S. cases, both survived, were recorded in May 2014.

In May 2015, an outbreak of MERS-CoV occurred in the Republic of Korea, when a man who had traveled to the Middle East, visited four hospitals in the Seoul area to treat his illness. This caused one of the largest outbreaks of MERS-CoV outside the Middle East. 

Coronavirus Disease 2019, COVID-19

In December 2019, a pneumonia outbreak was reported in Wuhan, China on December 31, 2019, the outbreak was traced to a novel strain of coronavirus, which was given the name 2019-nCoV by the WHO later renamed SARS-CoV-2 by the International Committee on Taxonomy of Viruses.

The Wuhan strain has been identified as a new strain of β-coronavirus from group 2B with approximately 70% genetic similarity to the SARS-CoV. The virus has a 96% similarity to a bat coronavirus, so it is widely suspected to originate from bats as well, new variants due to mutations have been identified since the begin.

Observation

-The 70% of genetic similarity to SARS-CoV, justifies for myself the reason of cross reactivity from previous infections with other types of coronaviruses, like those responsible of common cold, for example, and why some people were infected in more serious and dramatic way while others in a mild way, or not at all, or did not get sick despite of not being vaccinated; all of this to account to the distinction of the population in secretor and non-secretor, blood group influence and methylation status-

Sources:

Coronaviruses: Wikipedia

Coronaviruses Types: CDC

Picture by: shreddfigure.com

Thanks for Reading

Mariarosaria M.

The Biology and Microbiology of Coronaviruses

See the source image

Microbiology and Virology were two branches of Biology I was always very interested, and since the pandemic I have been dedicating more time to these discipline

With the raise of cases all over around the world due to new variants, mutations, and other reasons our interest and concerns for the coronaviruses have regrown, and for this reason I have wanted to look to a much deeper way at the microbiology of these viruses. I am not here to talk about all the matter of this pandemic because I am certainly not qualified for this and, honestly, in the middle of not knowing what to believe myself; either way I am not going to talk about remedies and therapies; there is plenty of information out there, the best specialists and scientists are greatly conversing and debating about from both sides, conventional and integrative doctors, those who believe in vaccine and those who not. I have been treating in previous blogs, and many of the natural adjuvants of support and prevention therapies look like to be still valid and the same.

I have watched among others the last of a serial of webinars on Covid-19 hosted by Dr. Michael Murray, one of the naturopaths I trust most in regard of this complicate and complex dilemma we all are living; it seems that he has revalidated the most of his believes and founds with few more updates on the whole situation and results from clinical trials. It can be found on YouTube for whom is interested.

Coronaviruses

Coronaviruses- as of Wikipedia description, one of my preferred and recurrent sources of information- are a group of RNA viruses responsible of diseases in mammals and birds. In humans and birds, they cause respiratory tract infections diseases that can range from mild to lethal. Mild illnesses in humans include the common cold also caused by other viruses like rhinoviruses, while more lethal varieties can cause SARSMERS, and COVID-19.  

Microbiology

Coronaviruses belong to the family of Coronaviridae and are enveloped viruses with a positive-sense single stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses is one of the largest among RNA viruses and has a 5’ methylated cap and a 3’polyadenylated tail. They have characteristic club-shaped spikes that project from their surface, which under electron microscope appears as an image of the solar corona, from which their name derives.

This class of viruses are large spherical particles with unique surface projections. Their size is highly variable with average diameters of 80 to 120 nm. Extreme sizes are known from 50 to 200 nm in diameter. The total molecular mass is on average 40,000 Dalton. They are enclosed in an envelope surrounded of various protein molecules. The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell.

The envelope and membrane protein are the structural proteins that combined with the lipid bilayer to form the viral envelope. Spike proteins are needed for interaction with the host cells. The membrane protein is the main structural protein of the envelope that provides the general shape. This protein is crucial during the assembly, growing, envelope formation, and pathogenesis stages of the virus lifecycle.

The envelope’s proteins are highly variable in different species. They are integral proteins and have two domains, a transmembrane domain and an extramembrane C-terminal domain. They are almost fully α-helical, with a single α-helical transmembrane domain, and form pentameric ionic channels in the lipid bilayer. They are responsible for virion assembly, intracellular operating, and morphogenesis.

The spikes, the most typical feature of coronaviruses, are responsible for the corona- or halo-like surface. A coronavirus particle on average is made of 74 surface spikes. Each spike is about 20 nm long and is composed of a trimer of the spike. The spike, or S protein is in turn composed of two subunits, S1 and S2.

The spike proteins are a class of fusion proteins which mediate the receptor binding and membrane fusion between the virus and host cell. The S1 subunit forms the head of the spike and has the receptor-binding domain. The S2 subunit secures the spike in the viral envelope and on protease activation enables fusion.

The two subunits remain noncovalently linked as they are exposed on the viral surface until they attach to the host cell membrane. In a functionally active state, three S1 are attached to two S2 subunits. The subunit complex is split into individual subunits when the virus binds and fuses with the host cell under the action of proteases like the cathepsin family and transmembrane protease serine 2 of the host cell.

S1 proteins are the most critical components in terms of infection. They are also the most variable components as they are responsible for host cell specificity. They possess two major domains, the N-terminal domain, and the C-terminal domain, both of which serve as the receptor-binding domains. The N-terminal domain recognizes and bind sugars on the surface of the host cell.

A subset of coronaviruses, specifically the members of beta coronavirus subgroup A, also has a shorter spike-like surface protein called hemagglutinin esterase. These proteins appear as tiny surface projections of 5 to 7 nm long implanted between the spikes. They play a role also in the attachment and detachment from the host cell.

Inside the envelope, there is the nucleocapsid, which is formed from multiple copies of the nucleocapsid protein, which are bound to the positive-sense single-stranded RNA genome in a continuous beads-on- a-string type conformation. Nucleocapsid protein is a phosphoprotein divided into three conserved domains. Most of the protein is made up of domains 1 and 2, which are typically rich in arginine and lysine while domain 3 has a short carboxy terminal end and has a net negative charge due to excess of acidic over basic amino acid residues.

-The chemical composition is what can influence the behavior and function of a molecule, specificity, site of attachment and more, and this is probably one of the characteristics that researchers study to try to find answers and solutions-

Replication Cycle

Cell Entry

Infection begins when the viral spike protein attaches to its complementary host cell receptor. After attachment, a protease of the host cell cuts and activates the receptor-attached spike protein. Depending on the host cell protease available, cleavage and activation allows the virus to enter the host cell by endocytosis or direct fusion of the viral envelope with the host membrane.

-This is where most of the drugs are, eventually, supposed to act to block the entrance of the virus into the cells-

Genome Translation

As into the host cell, the virus particle is uncoated, and its genome enters the cell cytoplasm. The coronavirus RNA genome has a 5′ methylated cap and a 3′ polyadenylated tail, which allows it to act like a messenger RNA, or m-RNA and be directly translated by the host cell’s ribosomes. The host ribosomes translate the initial overlapping open reading frame and a same frame of the virus genome into two large overlapping polyproteins.

The larger polyprotein is a result of a -1ribosomal frameshift caused by a slippery sequence (UUUAAAC) and a downstream RNA pseudoknot at the end of open reading frame.  The ribosomal frameshift allows for the continuous translation of the overlapping reading frames.

The polyproteins have their own proteases, which split the polyproteins at different specific sites. Product proteins include various replication proteins such as RNA-dependent RNA polymerase, (RdRp), RNA helicase, and exoribonuclease.

Replicase-Transcriptase

Several of the nonstructural proteins come together to form a multi-protein replicase-transcriptase complex. The main replicase-transcriptase protein is the RNA-dependent RNA polymerase which is directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease nonstructural protein provides extra reliability to replication by delivering a proofreading function which the RNA-dependent RNA polymerase lacks.

Replication

One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA.

Transcription

The other important function of the complex is to transcribe the viral genome. The replication process is followed by the transcription of these negative-sense subgenomic RNA molecules to their corresponding positive-sense mRNAs. The subgenomic mRNAs form a “nested set” which have a common 5′-head and partially duplicate 3′-end.

-I probably missed to interpret the nature of the coronaviruses transcriptase from the begin because I thought that the RNA-transcriptase were a reverse-transcriptase so as in Retroviruses, and that therefore they were producing DNA from RNA templates, but it does not look to be. In the same time I have read some articles from PubMed that were talking about the possibility of “reverse transcribed” of SARS-CoV-2RNA to explain the why of many positive cases and reinfection. I am going to discuss in a next blog-

Recombination

The replicase-transcriptase complex is also capable of genetic recombination when at least two viral genomes are present in the same infected cell. RNA recombination appears to be a major driving force in determining genetic variability within a coronavirus species, the capability of a coronavirus species to jump from one host to another and, infrequently, in determining the emergence of novel coronaviruses. The exact mechanism of recombination in coronaviruses is unclear, but likely involves template switching during genome replication.

Assembly and release

The replicated positive-sense genomic RNA becomes the genome of the progeny viruses. The mRNAs are gene transcripts of the last third of the virus genome after the initial overlapping reading frame. These mRNAs are translated by the host’s ribosomes into the structural proteins and many accessory proteins. RNA translation occurs inside the endoplasmic reticulum. The viral structural proteins S, E, and M move along the secretory pathway into the Golgi intermediate compartment. There, the M proteins direct most protein-protein interactions required for the assembly of viruses following its binding to the nucleocapsid. Progeny viruses are then released from the host cell by exocytosis through secretory vesicles. Once released the viruses can infect other host cells.

Transmission

Infected carriers can shed viruses into the environment. The interaction of the coronavirus spike protein with its complementary cell receptors is central in determining the tissue tropism, infectivity, and species range of the released virus. Coronaviruses mainly target epithelial cells. They are transmitted from one host to another host, depending on the coronavirus species, by either an aerosolcontaminated objects, or fecal-oral route.

Human coronaviruses infect the epithelial cells of the respiratory tract, while animal coronaviruses generally infect the epithelial cells of the digestive tract. SARS coronavirus, for example, infects the human epithelial cells of the lungs via an aerosol route by binding to the angiotensin-converting enzyme 2, (ACE2) receptors. Transmissible gastroenteritis coronavirus, which is an alfa-coronavirus, infects pigs’ epithelial cells of the digestive tract via a fecal-oral route by binding to the alanine amino-peptidase receptor.

Source:

Wikipedia, Coronavirus

Picture by jasminedirectory.com

To be continued

Thanks for Reading

Mariarosaria M.

Cholesterol Metabolism and Treatments of Hypercholesterolemia

See the source image

Regulation of Cholesterol

The cellular supply of cholesterol is maintained at a balanced level by three distinct mechanisms:

1)Regulation of HMGR, HMG-reductase activity and levels. HMG is the product of condensation of two molecules of Aceto-acetyl-coA in the biosynthesis of cholesterol.

2)Regulation of excess intracellular free cholesterol through the activity of sterol O-acyltransferases, SOAT1 and SOAT2 which is the predominant activity in liver.

3)Regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-mediated reverse transport.

The first three control mechanisms are exerted by cholesterol itself. Cholesterol acts as a feed-back inhibitor of pre-existing HMGR as well as inducing rapid degradation of the enzyme. In addition, when cholesterol is in excess the amount of mRNA for HMGR is reduced because of decreased expression of the gene.

-The feedback mechanisms are typical of metabolic pathways, same happens with thyroid hormones, T3 and T4, they act as inhibitors of their own production when levels are too high controlling the release of TSH whose demand decreases in this case and its level goes down; in the opposite way when levels of the hormones are low, there is more demand of production and TSH level goes up.

The thyroid metabolism also influences the cholesterol level, especially while in hypothyroidism, in this case with low levels of thyroid hormones all the metabolic functions are reduced and delayed and therefore there is not metabolization of cholesterol and more accumulation-

Regulation of HMGR through covalent modification occurs because of phosphorylation and dephosphorylation. The enzyme is most active in its unmodified form. Phosphorylation of the enzyme decreases its activity. HMGR is phosphorylated by AMP-activated protein-kinase, AMPK. AMPK itself is activated via phosphorylation. Phosphorylation of AMPK is catalyzed by at least two enzymes.

The activity of HMGR is additionally controlled by the cAMP signaling pathway. Increases in cAMP lead to activation of cAMP-dependent protein kinase, PKA. When the regulatory subunits are phosphorylated by PKA the activity of the associated phosphatases is reduced which results in AMPK remaining in the phosphorylated and active state, and HMGR in the phosphorylated and inactive state.

As the stimulus leading to increased cAMP production is removed, the level of phosphorylation decreases and that of dephosphorylations increases. The net result is a return to a higher level of HMGR activity. In contrast, insulin leads to a decrease in cAMP, which in turn activates cholesterol synthesis.

The ability of insulin to stimulate, and glucagon to inhibit HMGR activity is consistent with the effects of these hormones on other metabolic pathways. The basic function of these two hormones is to control the availability and delivery of energy to all cells of the body.

Long-term control of HMGR activity is exerted primarily through control over the synthesis and degradation of the enzyme. When levels of cholesterol are high, the level of expression of the HMGR gene is reduced. Conversely, reduced levels of cholesterol activate expression of the gene. Insulin also brings about long-term regulation of cholesterol metabolism by increasing the level of HMGR synthesis.

Treatment of Hypercholesterolemia

Reductions in circulating cholesterol levels can have profound positive impacts on cardiovascular disease, particularly on atherosclerosis.

Yet, the statement tests that: “Control of dietary intake is one of the easiest and least cost way to achieve reductions in cholesterol”.

-This assertion, and which is the obvious one contrasts the one stating that “More cholesterol we introduce with diet, less is produced by liver”. But, reasoning they seem both to make sense if we consider the feedback mechanism-

Recent studies in laboratory rats have demonstrated an additional benefit of reductions in dietary cholesterol intake. In these animals it was observed that reductions in dietary cholesterol not only resulted in decreased serum VLDL and LDL, and increased HDL but DNA synthesis was also shown to be increased in the thymus and spleen. Based on histological examination of the spleen, thymus, and lymph nodes it was found that there was an increased number of immature cells and enhanced mitotic activity indicative of enhanced proliferation. These results suggest that a marked reduction in serum LDL, induced by reduced cholesterol intake, stimulates enhanced DNA synthesis and cell proliferation.

Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily pointed at reducing the risk of atherosclerosis and subsequent coronary artery disease that occurs in patients with elevated circulating lipids. Drug therapy usually is considered as an option only if non-pharmacologic interventions like altered diet and exercise have failed to lower plasma lipids.

Statins, the most common therapy, are drugs that inhibits HMG-CoA reductase, HMGR. The net result of treatment is an increased cellular uptake of LDL, since the intracellular synthesis of cholesterol is inhibited, and cells are therefore dependent on extracellular sources of cholesterol. However, since mevalonate, the product of the HMG-CoA reductase reaction, is required for the synthesis of other important isoprenoid compounds besides cholesterol, long-term treatments bring some risk of toxicity.

It seems that the statins have become recognized as a class of drugs capable of more pharmacologic benefits than just lowering blood cholesterol levels via their actions on HMGR. Part of the cardiac benefit of the statins relates to their ability to regulate the production of anti-inflammatory compounds.

Statins seems to also have some effects on immune function, like attenuation of autoimmune disease, inhibition of T-cell proliferation, inhibition of inflammatory co-stimulatory molecule expression, decreases in leukocyte infiltration, and promotion of a shift in cytokine profiles of helper T-cell types from Th1 to Th2.

Another common drug used now for lowering cholesterol is Nicotinic acid, or Niacin, or vitamin B3. This reduces the plasma levels of both VLDL and LDL by inhibiting hepatic VLDL secretion. In addition, nicotinic acid administration strongly increases the circulating levels of HDL. Unfortunately, is administration is compromised for the unpleasant side-effect of flushing, as strong cutaneous vasodilation.

-These the most common drugs of conventional therapy, but there are more than these-

Alternative Options of Treatments

The natural options for lowering cholesterol are various, I will just mention the most typical elements present in the major number of supplements, and most typical suggestions and tips that can be found online as soon as we look for “natural option, or natural remedies for lowering cholesterol”.

As we all know, diet and exercise are number one priority and essential steps to take for most of the diseases, and fibers, especially soluble fibers, are the most recommended, oat, barley and psyllium are the best, but oat-B-glucans are a specific type of oat that seems to lower the bad cholesterol at particularly good percentage of rate.

Here some of the most typical suggested foods to lower cholesterol.

By Pinterest.com

Niacin, fish oil, CoQ10, garlic, plant sterols and stanols, red yeast rice (the natural version of statins), pantetheine or vitamin B5 analog, olive leaves, bergamot, berberine, choline and phosphatidylcholine, and many more compounds or herbs can be found in different arrangements and assembling in supplements for cholesterol; it depends on the company, some they have more elements, some usually two or three of these mentioned.

What it looks important about mono and polyunsaturated fats, and which are respectively oil of olive and omega’s-3 is that they reduce the oxidation of lipoprotein, the dangerous step toward plaque formation and clogging of arteries, decrease of LDL, and increasing of HDL.

One of the most recommended products online besides the most typical is “Cholesteoff”, made by Nature Made and containing Sterols and Stanols, plus Pantetheine, I believe. I am mentioning because I found some interesting information in regard of soy and so of sterols and seems that they really have been taking always more in consideration.  

In this paragraph from a book of Natural Medicine has been examined the influences of soy on cholesterol metabolism; it seems that regular intake of soy foods, especially in place of more common animal protein sources, leads to modest meaningful cholesterol reductions in hypercholesterolemic individuals. Soy’s main mechanisms of action for this appear to be interfering with cholesterol absorption, increasing fecal bile acids excretion, and upregulating low density lipoprotein (LDL) receptor expression.

Individual studies found that hyperlipidemic men and women receiving soy protein with isoflavones in addition to a National Cholesterol Education Program showed significantly lower total cholesterol, estimated coronary artery disease risk, total/high-density lipoprotein (HDL) ratio, LDL/HDL ratio, and apolipoprotein B/A-1 ratio than those using the program and diet alone; LDL cholesterol, apolipoprotein B,, homocysteine, and oxidized LDL levels were also lower with soy than during the program and diet alone.

In a similar study hypercholesterolemic postmenopausal women receiving soy protein and isoflavones in addition to program and diets showed a greater decrease in non-HDL cholesterol than those receiving program and diets alone, and those receiving soy protein and isoflavones also showed increases in HDL cholesterol, as well as decrease in LDL levels.

The results of a 2010 study suggest that soy foods’ potential benefits to lipid profiles may be significantly improved by combination with a prebiotic like inulin. Hyperlipidemic men and women given 30 g soy protein, 61 mg soy isoflavones, and 10 g oligofructose-enriched inulin showed significant improvements in measures of LDL and HDL cholesterol compared with either soy or prebiotic alone.

As for the most today the solutions are different within the same conventional medicine and with the integrative even more; it is always up to us the final decision and what we feel are body can tolerate or not, interferences with other medications or supplements that we already take and capability of the systems to process everything, mainly gastroenteric and urinary system and even more the liver where everything is processed.

Thanks For Reading.

Mariarosaria M.

Sources:

Themedicalbiochemistrypage.org

Textbook of Natural Medicine, 2020, Cheryl Kos ND, by sciencedirect.com

Introductory picture by Healthline.com

Cholesterol, Metabolic Pathways and Correlations

Cholesterol is one of the subjects I am personally interested because despite of my lifestyle and the way I eat, it keeps on average normally high, especially the LDL compound.

As much as I am acknowledged and understand as biologist, I have learned from books and articles, talked about in previous blogs, and aware of the benefits and function, I am honestly, at this time concerned about, especially in regard of the LDL compound, and this is why I have been researching more and looked at a molecular and biochemical level trying to understand where this cholesterol comes from, and/or which pathways are involved and connected, as much as  always more persuaded of the genetic origins and overproduction by liver.

I still cannot get the complete picture of what does happen inside the incredible microscopic environment of our body and our cells and systems, so interconnected and complex, especially when we start to look at the metabolic pathways.

The questions remain for me a little bit, and this is the reason why I am brainstorming and assembling fractions of these biochemical steps and descriptions partially rearranged to simplify the original content and clarified.

Cholesterol is a sterol, present in animal tissue, in large concentration in liver and brain. It is the structural component of all cell membranes, regulates fluidity and confers stability, and has an important role in brain synapsis and the immune system.

It is also the precursors of bile acids, steroid hormones, and vitamin D.

Hydrophobic compound made of four fused hydrocarbon rings, A, B, C and D called steroid nucleus, and with 8 carbon branched hydrocarbon chain (CH) attached to C17 of D-ring. Ring-A has a hydroxyl group (OH) at C3, ring-B a double bond between C5 and C6.

Cholesterol is an extremely important biological molecule that has roles in membrane structure as well as a precursor for the synthesis of the steroid hormones, bile acids, and vitamin D. Both dietary cholesterol, and that synthesized “de novo”, are transported through the circulation in lipoprotein particles. The same is true of cholesteryl esters, the form in which cholesterol is stored in cells. Due to its important role in membrane function, all cells express the enzymes of cholesterol biosynthesis.

The synthesis and utilization of cholesterol must be tightly regulated to prevent over-accumulation and abnormal deposition within the body. Of clinical importance is the abnormal deposition of cholesterol and cholesterol-rich lipoprotein in the coronary arteries, leading cause of atherosclerosis, and coronary arteries diseases.

Cholesterol Biosynthesis

Cholesterol is synthesized by all tissues in human, an essential molecule in many animals, including human but is not required in diet as all cells can synthesize it from simple precursors. It is made of 27 carbon compounds. All the carbon atoms in the cholesterol are provided by acetate. NADPH provides the reducing equivalents.

The biosynthesis pathway of cholesterol is endergonic and so requires ATP. To produce 1 mole of cholesterol are necessary 18 moles of Acetyl coA, 36 moles of ATP and 16 moles of NADPH.

In the first step in cholesterol biosynthesis, step I, two molecules of acetylcoA condenses to form AcetoacetylcoA. The reaction is catalyzed by enzyme thiolase.

AcetoacetylcoA condenses with another molecule of acetylcoA to form β-hydroxyl-β-methyl-glutaryl-coA (HMG).

-An elementary observation, acetyl-coA is a molecule of the Krebs cycle, the major step of the general metabolism where glucose in presence of oxygen is metabolized and decomposed in water and carbon dioxide through a number of reactions catalyzed by different enzymes and with ox-reduction processes that allow flow of electrons and release of energy. The cholesterol biosynthesis therefore steels substrates for the Krebs cycle, generating increase demand for glucose and so of carbohydrates and that is why also the carbs relate to the cholesterol production-

In step II, reduction of HMG-coA to Mevalonate is catalyzed by HMG-coA reductase enzyme and happens in in cytosol, in this reaction 2 NADPH are used for donation of 2 electrons. This reaction is the key step in regulation of cholesterol biosynthesis.

In step III, three phosphate groups are transferred from three ATP molecules to Mevalonate to generate Isoprene units.

During step IV, six activated units of isoprene condense with elimination of both pyrophosphate group to form Squalene.

In step V Squalene is converted in Lanosterol, which contains four rings steroid nucleus, through hydroxylation and cyclization utilizing NADHP.

Finally in Step VI happens the conversion of Lanosterol into Cholesterol where Lanosterol undergoes a series of about 20 reactions to finally convert into cholesterol.

Cholesterol Synthesis Pathway - YouTube

-These are instead some and more common molecules involved in the pathways, the steps are endless and too complex to report each of them, I have tried to summarize in a way –

Cytochrome P450 enzymes are involved in a diverse array of biological processes that includes lipid, cholesterol, and steroid metabolism. The common nomenclature for P450 enzymes is CYP. There are at least 57 CYP enzymes in human tissues, eight of these are involved in cholesterol biosynthesis and metabolism, which includes conversion of cholesterol to bile acids. CYP metabolism of cholesterol generates several oxysterols that function as biologically active molecules such as in the activation of liver receptors.

Coenzyme Q, or ubiquinone is a red-ox active molecule that is composed of a benzoquinone ring conjugated to a polyisoprenoid tail that is of variable length in different species and organisms. In humans the polyisoprenoid tail consists of 10 isoprenoid units which impart the common name for the molecule as CoQ10. In undergoing reduction and oxidation reactions the electrons are accepted and donated from benzoquinone ring. The polyisoprenoid tail of ubiquinone serves to anchor the molecule in the membrane.

-Here is why the importance of taking CoQ10 during therapy with Statins, as we can see CoQ10 is an important molecule in the cholesterol metabolism-

Heme A is an essential component of the oxidative phosphorylation pathway by serving as the prosthetic group for cytochrome c oxidase of complex IV. Cytochrome c is so-called due to the presence of two distinct heme a prosthetic group with heme a being the direct electron donor in the complex IV catalyzed reduction of O2 to H2O.

Regulation of Cholesterol

A relatively constant level of cholesterol in the blood (150–200 mg/dL) is maintained primarily by controlling the level of de novo synthesis. The level of cholesterol synthesis is regulated in part by the dietary intake of cholesterol.

-In regard of this assertion I just want to mention some that I read from a nutrition book, and that made me wonder, the book was stating that “more fat we eat, less cholesterol is produced from the body” Is this the reason why they were emphasizing the importance of eating fats and of ketogenic diet? To me sounds still controversial-

Cholesterol from both diet and synthesis is utilized in the formation of membranes and in the synthesis of the steroid hormones and bile acids. The greatest proportion of cholesterol is used in bile acid synthesis.

The cellular supply of cholesterol is maintained at a steady level by three distinct mechanisms:

  1. Regulation of HMGR activity and levels.
  2. Regulation of excess intracellular free cholesterol through the activity of sterol O-acyltransferases, SOAT1 and SOAT2 which is the predominant activity in liver.
  3. Regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-mediated reverse transport.

Regulation of HMGR activity is the primary means for controlling the level of cholesterol biosynthesis. The enzyme is controlled by four distinct mechanisms: feed-back inhibition, control of gene expression, rate of enzyme degradation and phosphorylation-dephosphorylation.

The Utilization of Cholesterol

Cholesterol is transported in the plasma predominantly as cholesteryl ester associated with lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDL. The liver synthesizes VLDL, and these are converted to LDL through the action of endothelial cell-associated lipoprotein lipase. Cholesterol found in plasma membranes can be extracted by HDL and esterified by the HDL-associated enzyme lecithin-cholesterol acyltransferase, LCAT. The cholesterol acquired from peripheral tissues by HDL can then be transferred to VLDL and LDL via the action of cholesteryl ester transfer protein (CETP) which is associated with HDL.

Reverse cholesterol transport allows peripheral cholesterol to be returned to the liver in LDL. Ultimately, cholesterol is excreted in the bile as free cholesterol or as bile salts following conversion to bile acids in the liver.

-To make simple the role of HDL is to absorb cholesterol from the body and to carry to the liver from where then will be flushed out from the excretory systems, while LDL delivers fat molecules to the cells and is involved in atherosclerosis, a process where LDL is oxidized within the arterial walls and responsible of plaques production and so of blood flow blockage and cardiovascular consequences-

As we can see the steps and regulation are endless, and utilization differs based on the lipoproteins of transport and site of production, for this reason the subject must be separated in more steps.

To be continued

Thanks For Reading

Mariarosaria M.

Sources:

Pubchem.ncbi.nih.gov/pathway/wikipathways

themedicalbiochemistrypage.org

Onlinebiologynotes.com

Picture by youtube.com