Protein Structure Animation

Proteins are an important class of biological macromolecules present in all biological organisms, made up of such elements as carbon, hydrogen, nitrogen, phosphorous, oxygen, and sulfur. All proteins are polymers of amino acids. The polymers, also known as polypeptides consist of a sequence of 20 different L-α-amino acids, also referred to as residues. For chains under 40 residues the term peptide is frequently used instead of protein. To be able to perform their biological function, proteins fold into one, or more, specific spatial conformations, driven by a number of noncovalent interactions such as hydrogen bonding, ionic interactions, Van der Waals' forces and hydrophobic packing. In order to understand the functions of proteins at a molecular level, it is often necessary to determine the three dimensional structure of proteins. This is the topic of the scientific field of structural biology, that employs techniques such as X-ray crystallography or NMR spectroscopy, to determine the structure of proteins.


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A number of residues is necessary to perform a particular biochemical function, and around 40-50 residues appears to be the lower limit for a functional domain size. Protein sizes range from this lower limit to several thousand residues in multi-functional or structural proteins. However, the current estimate for the average protein length is around 300 residues. Very large aggregates can be formed from protein subunits, for example many thousand actin molecules assemble into a collagen filament.

Biochemistry refers to four distinct aspects of a protein's structure:
  • Primary structure - the amino acid sequence of the peptide chains.
  • Secondary structure - highly regular sub-structures (alpha helix and strands of beta sheet) which are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule.
  • Tertiary structure - Three-dimensional structure of a single protein molecule; a spatial arrangement of the secondary structures.
  • Quaternary structure - complex of several protein molecules or polypeptide chains, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

In addition to these levels of structure, a protein may shift between several similar structures in performing its biological function. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as chemical conformation, and transitions between them are called conformational changes.

The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. These peptide bonds provide rigidity to the protein. The two ends of the amino acid chain are referred to as the C-terminal end or carboxyl terminus (C-terminus) and the N-terminal end or amino terminus (N-terminus) based on the nature of the free group on each extremity.

The various types of secondary structure are defined by their patterns of hydrogen bonds between the main-chain peptide groups. However, these hydrogen bonds are generally not stable by themselves, since the water-amide hydrogen bond is generally more favorable than the amide-amide hydrogen bond. Thus, secondary structure is stable only when the local concentration of water is sufficiently low, e.g., in the molten globule or fully folded states.

Similarly, the formation of molten globules and tertiary structure is driven mainly by structurally non-specific interactions, such as the rough propensities of the amino acids and hydrophobic interactions. However, the tertiary structure is fixed only when the parts of a protein domain are locked into place by structurally specific interactions, such as ionic interactions (salt bridges), hydrogen bonds and the tight packing of side chains. The tertiary structure of extracellular proteins can also be stabilized by disulfide bonds, which reduce the entropy of the unfolded state; disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment.

Primary structure

The sequence of the different amino acids is called the primary structure of the peptide or protein. Counting of residues always starts at the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. The primary structure of a protein is determined by the gene corresponding to the protein. A specific sequence of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a protein is unique to that protein, and defines the structure and function of the protein. The sequence of a protein can be determined by methods such as Edman degradation or tandem mass spectrometry. Often however, it is read directly from the sequence of the gene using the genetic code. Post-transcriptional modifications such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene.

Secondary structure
By building models of peptides using known information about bond lengths and angles, the first elements of secondary structure, the alpha helix and the beta sheet, were suggested in 1951 by Linus Pauling and coworkers.Both the alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. These secondary structure elements only depend on properties that all the residues have in common, explaining why they occur frequently in most proteins. Since then other elements of secondary structure have been discovered such as various loops and other forms of helices. The part of the backbone that is not in a regular secondary structure is said to be random coil. Each of these two secondary structure elements have a regular geometry, meaning they are constrained to specific values of the dihedral angles ψ and φ. Thus they can be found in a specific region of the Ramachandran plot.

Tertiary structure

The elements of secondary structure are usually folded into a compact shape using a variety of loops and turns. The formation of tertiary structure is usually driven by the burial of hydrophobic residues, but other interactions such as hydrogen bonding, ionic interactions and disulfide bonds can also stabilize the tertiary structure. The tertiary structure encompasses all the noncovalent interactions that are not considered secondary structure, and is what defines the overall fold of the protein, and is usually indispensable for the function of the protein.

Quaternary structure

The quaternary structure is the interaction between several chains of peptide bonds. The individual chains are called subunits. The individual subunits are not necessarily covalently connected, but might be connected by a disulfide bond. Not all proteins have quaternary structure, since they might be functional as monomers. The quaternary structure is stabilized by the same range of interactions as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. Multimers made up of identical subunits may be referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits may be referred to with a prefix of "hetero-" (e.g. a heterodimer). Tertiary structures vary greatly from one protein to another. They are held together by glycosydic and covalent bonds.

Side chain conformation

The atoms along the side chain are named with Greek letters in Greek alphabetical order: α, β, γ, δ, є and so on. Cα refers to the carbon atom closest to the carbonyl group of that amino acid, Cβ the second closest and so on. The Cα is usually considered a part of the backbone. The dihedral angles around the bonds between these atoms are named χ1, χ2, χ3 etc. E.g. the first and second carbon atom in the side chain of lysine is named α and β, and the dihedral angle around the α-β bond is named χ1. Side chains can be in different conformations called gauche(-), trans and gauche(+). Side chains generally tend to try to come into a staggered conformation around χ2, driven by the minimization of the overlap between the electron orbitals of the hydrogen atoms.


Angiogenesis is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state.


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Sprouting angiogenesis was the first identified form of angiogenesis. It occurs in several well-characterized stages. First, biological signals known as angiogenic growth factors activate receptors present on endothelial cells present in pre-existing venular blood vessels. Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basement membrane in order to allow endothelial cells to escape from the original (parent) vessel walls. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelial cells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, called integrins. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels.

Types of angiogenesis

Sprouting angiogenesis
Intussusceptive angiogenesis

Intussusception, also known as splitting angiogenesis, was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis. First, the two opposing capillary walls establish a zone of contact. Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen. Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen. Finally, the core is fleshed out with no alterations to the basic structure. Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.

Modern Terminology of Angiogenesis

Besides the differentiation between “Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists the today more common differentiation between the following types of angiogenesis:

Vasculogenesis – Formation of vascular structures from circulating or tissue-resident endothelial stem cells (angioblasts), which proliferate into de-novo endothelial cells. This form particularly relates to the embryonal development of the vascular system.

Angiogenesis – Formation of thin-walled endothelium-lined structures with/without muscular smooth muscle wall and pericytes (fibrocytes). This form plays an important role during the adult life span, also as "repair mechanism" of damaged tissues.

Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.

Because it turned out that even this differentiation is not a sharp one, today quite often the term “Angiogenesis” is used summarizing all different types and modifications of arterial vessel growth.

Therapeutic angiogenesis

Therapeutic angiogenesis is the application of specific compounds which may inhibit or induce the creation of new blood vessels in the body in order to combat disease. The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may retard repair or some other function. Several diseases (eg. ischemic chronic wounds) are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.

The modern clinical application of the principle “angiogenesis” can be divided into two main areas: 1. Anti-angiogenic therapies (historically, research started with); 2. Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying to fight cancer and malignancies(because tumors, in general, are nutrition- and oxygen-dependent, thus being in need of adequate blood supply), the pro-angiogenic therapies are becoming more and more important in the search of new treatment options for cardiovascular diseases (the number one cause of death in the Western world). One of the world-wide first applications of usage of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease. Today, clinical research is ongoing in various clinical trials to promote therapeutic angiogenesis for a variety of atherosclerotic diseases, like coronary heart disease, peripheral arterial disease, wound healing disorders, etc.

Also, regarding the “mode of action”, pro-angiogenic methods can be differentiated into three main categories: 1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based therapies.

There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells; 2. Risk of an undesired immune response; 3 Potential toxicity, immunogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases. In contrast, pro-angiogenic protein therapy uses well defined, precisely structured proteins, with previously defined optimal doses of the individual protein for disease states, and with well-known biological effects. On the other hand, an obstacle of protein therapy is the mode of delivery: oral, intravenous, intra-arterial, or intramuscular routes of the protein’s administration are not always as effective as desired; the therapeutic protein can be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still in an early stage of research – with many open questions regarding best cell types and dosages to use.

The fibroblast growth factor (FGF) family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic FGF) consists to date of at least 22 known members. Most are 16-18 kDa single chain peptides and display high affinity to heparin and heparan sulfate. In general, FGFs stimulate a variety of cellular functions by binding to cell surface FGF-receptors in the presence of heparin proteoglycans. The FGF-receptor family is comprised of seven members and all the receptor proteins are single chain receptor tyrosine kinases that become activated through autophosphorylation induced by a mechanism of FGF mediated receptor dimerization. Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses, including cell differentiation, proliferation, and matrix dissolution – thus initiating a process of mitogenic activity critical for the growth of endothelial cells, fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members of the FGF family, can bind to all seven FGF-receptor subtypes, making it the broadest acting member of the FGF family, and a potent mitogen for the diverse cell types needed to mount an angiogenic response in damaged (hypoxic) tissues, where up regulation of FGF-receptors occurs. FGF-1 stimulates the proliferation and differentiation of all cell types necessary for building an arterial vessel, including endothelial cells and smooth muscle cells; this fact distinguishes FGF-1 from other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) which primarily drives the formation of new capillaries.

Until now (2007), three human clinical trials have been successfully completed with FGF-1 in which the angiogenic protein was injected directly into the damaged heart muscle. Also, one additional human FGF-1 trial has been completed to promote wound healing in diabetics with chronic wounds.

Besides FGF-1, one of the most important functions of also fibroblast growth factor-2 (FGF-2 or bFGF) is the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube-like structures, thus promoting angiogenesis. FGF-2 is a more potent angiogenic factor than VEGF or PDGF (platelet-derived growth factor), however, less potent than FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are important players in wound healing. They stimulate the proliferation of fibroblasts and endothelial cells that give rise to angiogenesis and developing granulation tissue, both increase blood supply and fill up a wound space/cavity early in the wound healing process.


VEGF (Vascular Endothelial Growth Factor) has been demonstrated to be a major contributor to angiogenesis, increasing the number of capillaries in a given network. Initial in vitro studies demonstrated that bovine capillary endothelial cells will proliferate and show signs of tube structures upon stimulation by VEGF and bFGF, although the results were more pronounced with VEGF. Upregulation of VEGF is a major component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment in vascular injuries. In vitro studies clearly demonstrate that VEGF is a potent stimulator of angiogenesis because in the presence of this growth factor plated endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries. VEGF causes a massive signaling cascade in endothelial cells. Binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producting NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. Mechanically, VEGF is upregulated with muscle contractions as a result of increased blood flow to affected areas. The increased flow also causes a large increase in the mRNA production of VEGF receptors 1 and 2. The increase in receptor production means that muscle contractions could cause upregulation of the signaling cascade relating to angiogenesis. As part of the angiogenic signaling cascade, NO is widely considered to be a major contributor to the angiogenic response because inhibition of NO significantly reduces the effects of angiogenic growth factors. However, inhibition of NO during exercise does not inhibit angiogenesis indicating that there are other factors involved in the angiogenic response.


The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies . Ang1 and Ang2 are protein growth factors which act by binding their receptors, Tie-1 and Tie-2; while this is somewhat controversial, it seems that cell signals are transmitted mostly by Tie-2; though some papers show physiologic signaling via Tie-1 as well. These receptors are tyrosine kinases. Thus, they can initiate cell signaling when ligand binding causes a dimerization that initiates phosphorylation on key tyrosines.


Another major contributor to angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries. These enzymes are highly regulated during the vessel formation process because destruction of the extracellular matrix would decrease the integrity of the microvasculature.

Applications of Angiogenesis
Tumor angiogenesis

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A tumor consists of a population of rapidly dividing and growing cancer cells. Mutations rapidly accrue within the population. These mutations (variation) allow the cancer cells (or sub-populations of cancer cells within a tumor) to develop drug resistance and escape therapy. Tumors cannot grow beyond a certain size, generally 1-2 mm³, due to a lack of oxygen and other essential nutrients.

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. Vascular Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients -- allowing for tumor expansion. On 18 July 2007 it was discovered that cancerous cells stop producing the anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG apparently limits beta-catenin which solicits angiogenesis.Other clinicians believe that angiogenesis really serves as a waste pathway, taking away the biological end products put out by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor. Evidence now suggests that the blood vessel in a given solid tumor may in fact be mosaic vessels, comprised of endothelial cells and tumor cells. This mosaicity allows for substantial shedding of tumor cells into the vasculature. The subsequent growth of such metastases will also require a supply of nutrients and oxygen or a waste disposal pathway.

Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire 'drug resistance' to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them. Recent studies by Klagsbrun, et al. have shown, however, that endothelial cells growing within tumors do carry genetic abnormalities. Thus, tumor vessels have the theoretical potential for developing acquired resistance to drugs. This is a new area of angiogenesis research being actively pursued.

Angiogenesis research is a cutting edge field in cancer research, and recent evidence also suggests that traditional therapies, such as radiation therapy, may actually work in part by targeting the genomically stable endothelial cell compartment, rather than the genomically unstable tumor cell compartment. New blood vessel formation is a relatively fragile process, subject to disruptive interference at several levels. In short, the therapy is the selection agent which is being used to kill a cell compartment. Tumor cells evolve resistance rapidly due to rapid generation time (days) and genomic instability (variation), whereas endothelial cells are a good target because of a long generation time (months) and genomic stability (low variation).

This is an example of selection in action at the cellular level, using a selection pressure to target and differentiate between varying populations of cells. The end result is the extinction of one species or population of cells (endothelial cells), followed by the collapse of the ecosystem (the tumor) due either to nutrient deprivation or self-pollution from the destruction of necessary waste pathways.

Angiogenesis-based tumour therapy relies on natural and synthetic angiogenesis inhibitors like angiostatin, endostatin and tumstatin. These are proteins that mainly originate as specific fragments pre-existing structural proteins like collagen or plasminogen.

Recently, the 1st FDA-approved therapy targeted at angiogenesis in cancer came on the market in the US. This is a monoclonal antibody directed against an isoform of VEGF. The commercial name of this antibody is Avastin, and the therapy has been approved for use in colorectal cancer in combination with established chemotherapy.

Angiogenesis for cardiovascular disease

Angiogenesis represents an excellent therapeutic target for the treatment of cardiovascular disease. It is a potent, physiological process that underlies the natural manner in which our bodies respond to a diminution of blood supply to vital organs, namely the production of new collateral vessels to overcome the ischemic insult. Perhaps the greatest reason for these trials’ failure to achieve success is the high occurrence of the “placebo effect” in studies employing treadmill exercise test readout. Thus, even though a majority of the treated patients in these trials experience relief of such clinical symptoms such as chest pain (angina), and generally performed better on most efficacy readouts, there were enough “responders” in the blinded placebo groups to render the trial inconclusive. In addition to the placebo effect, more recent animal studies have also highlighted various factors that may inhibit an angiogenesis response including certain drugs, smoking, and hypercholesterolemia.

Although shown to be relatively safe therapies, not one angiogenic therapeutic has yet made it through the gauntlet of clinical testing required for drug approval. By capitalizing on the large database of what did and did not work in previous clinical trials, results from more recent studies with redesigned clinical protocols give renewed hope that angiogenesis therapy will be a treatment choice for sufferers of cardiovascular disease resulting from occluded and/or stenotic vessels.

Early clinical studies with protein-based therapeutics largely focused on the intravenous or intracoronary administration of a particular growth factor to stimulate angiogenesis in the affected tissue or organ. Most of these trials did not achieve statistically significant improvements in their clinical endpoints. This ultimately led to an abandonment of this approach and a widespread belief in the field that protein therapy, especially with a single agent, was not a viable option to treat ischemic cardiovascular disease. However, the failure of gene- or cell-based therapy to deliver, as of yet, a suitable treatment choice for diseases resulting from poor blood flow, has led to a resurgence of interest in returning to protein-based therapy to stimulate angiogenesis. Lessons learned from earlier protein-based studies, which indicated that intravenous or intracoronary delivery of the protein was not efficacious, have led to completed and ongoing clinical trials in which the angiogenic protein is injected directly into the beating ischemic heart.

Such localized administration of the potent angiogenic growth factor, human FGF-1, has recently given promising results in clinical trials in no-option heart patients. Angiogenesis was documented by angiographically visible “blushing”, and functional exercise tests were also performed on a subset of patients. The attractiveness of protein therapy is that large amounts of the therapeutic agent can be injected into the ischemic area of interest, to pharmacologically start the process of blood vessel growth and collateral arteries’ formation. In addition, from pharmacokinetic data collected from the recent FGF-1 studies in the human heart, it appears that FGF-1, once it exits the heart is cleared in less than three hours from the circulation. This would presumably prevent FGF-1 from stimulating unwanted angiogenesis in other tissues of the bodies where it could potentially cause harm, such as the retina and in the kidneys. No serious adverse events have yet to be noted in any of the completed or ongoing clinical trials in which the FGF-1 protein is utilized as the therapeutic agent tom stimulate angiogenesis


Apoptosis (pronounced ă-pŏp-tŏ’sĭs) is a form of programmed cell death in multicellular organisms. It is one of the main types of programmed cell death (PCD) and involves a series of biochemical events leading to a characteristic cell morphology and death, in more specific terms, a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation (1-4). Processes of disposal of cellular debris whose results do not damage the organism differentiates apoptosis from necrosis.

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In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis, in general, confers advantages during an organism's life cycle. For example, the differentiation of fingers and toes in a developing human embryo occurs because cells between the fingers apoptose; the result is that the digits are separate. Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual's body weight.

Research on apoptosis has increased substantially since the early 1990s. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.

The process of apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers). Extracellular signals may include hormones, growth factors, nitric oxide or cytokines, and therefore must either cross the plasma membrane or transduce to effect a response. These signals may positively or negatively induce apoptosis; in this context the binding and subsequent initiation of apoptosis by a molecule is termed positive, whereas the active repression of apoptosis by a molecule is termed negative.

Intracellular apoptotic signalling is a response initiated by a cell in response to stress, and may ultimately result in cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, and hypoxia are all factors that can lead to the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.

Before the actual process of cell death is carried out by enzymes, apoptotic signals must be connected to the actual death pathway by way of regulatory proteins. This step allows apoptotic signals to either culminate in cell death, or be aborted should the cell no longer need to die. Several proteins are involved, however two main methods of achieving regulation have been identified; targeting mitochondria functionality, or directly transducing the signal via adapter proteins to the apoptotic mechanisms. The whole preparation process requires energy and functioning cell machinery.

Mitochondrial regulation

The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies - a fact exploited by some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways; they may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.There is also a growing body of evidence that indicates that nitric oxide (NO) is able to induce apoptosis by helping to dissipate the membrane potential of mitochondria and therefore make it more permeable.

Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases) are released into the cytosol following an increase in permeability. SMAC binds to inhibitor of apoptosis proteins (IAPs) and deactivates them, preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed. IAP also normally suppresses the activity of a group of cysteine proteases called caspases, which carry out the degradation of the cell, therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.

Cytochrome c is also released from mitochondria due to formation of a channel, MAC, in the outer mitochondrial membrane, and serves a regulatory function as it precedes morphological change associated with apoptosis. Once cytochrome c is released it binds with Apaf-1 and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3.

MAC is itself subject to regulation by various proteins, such as those encoded by the mammalian Bcl-2 family of anti-apoptopic genes, the homologs of the ced-9 gene found in C. elegans. Bcl-2 proteins are able to promote or inhibit apoptosis either by direct action on MAC or indirectly through other proteins. It is important to note that the actions of some Bcl-2 proteins are able to halt apoptosis even if cytochrome c has been released by the mitochondria.

Direct signal transduction
Two important examples of the direct initiation of apoptotic mechanisms in mammals include the TNF-induced (tumour necrosis factor) model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family coupled to extrinsic signals.

TNF is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of apoptosis. Most cells in the human body have two receptors for TNF: TNF-R1 and TNF-R2. The binding of TNF to TNF-R1 has been shown to initiate the pathway that leads to caspase activation via the intermediate membrane proteins TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses.The link between TNF and apoptosis shows why an abnormal production of TNF plays a fundamental role in several human diseases, especially in autoimmune diseases.

The Fas receptor (also known as Apo-1 or CD95) binds the Fas ligand (FasL), a transmembrane protein part of the TNF family.The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis. In other types of cells (type II), the Fas-DISC starts a feedback loop that spirals into increasing release of pro-apoptotic factors from mitochondria and the amplified activation of caspase-8.

Following TNF-R1 and Fas activation in mammalian cells a balance between pro-apoptotic (BAX,BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family is established. This balance is the proportion of pro-apoptotic homodimers that form in the outer-membrane of the mitochondrion. The pro-apoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of pro-apoptotic proteins under normal cell conditions of non-apoptotic cells is incompletely understood, but it has been found that a mitochondrial outer-membrane protein, VDAC2, interacts with BAK to keep this potentially-lethal apoptotic effector under control.When the death signal is received, products of the activation cascade displace VDAC2 and BAK is able to be activated.


Although many pathways and signals lead to apoptosis, there is only one mechanism that actually causes the death of the cell in this process; after the appropriate stimulus has been received by the cell and the necessary controls exerted, a cell will undergo the organised degradation of cellular organelles by activated proteolytic caspases. A cell undergoing apoptosis shows a characteristic morphology that can be observed with a microscope:

  1. Cell shrinkage and rounding due to the breakdown of the proteinaceous cytoskeleton by caspases.
  2. The cytoplasm appears dense, and the organelles appear tightly packed.
  3. Chromatin undergoes condensation into compact patches against the nuclear envelope in a process known as pyknosis, a hallmark of apoptosis.
  4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA.
  5. The cell membrane shows irregular buds known as blebs.
  6. The cell breaks apart into several vesicles called apoptotic bodies, which are then phagocytosed.

Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size. These give a characteristic "laddered" appearance on agar gel after electrophoresis. Tests for DNA laddering differentiate apoptosis from ischemic or toxic cell death.

Removal of dead cells

Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine, on their cell surface.Phosphatidylserine is normally found on the cytosolic surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a hypothetical protein known as scramblase. These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages.Upon recognition, the phagocyte reorganizes its cytoskeleton for engulfment of the cell. The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response.

Hepatitis C Animation

The Hepatitis C virus (HCV) is a small (50 nm in size), enveloped, single-stranded, positive sense RNA virus in the family Flaviviridae. Although hepatitis A, hepatitis B, and hepatitis C have similar names (because they all cause liver inflammation), these are distinctly different viruses both genetically and clinically.

Hepatitis C virus has a positive sense RNA genome that consists of a single open reading frame of 9600 nucleoside bases. At the 5’ and 3’ ends of the RNA there are regions , (UTR), that are not translated into proteins but are important to translation and replication of the viral RNA. The 5’ UTR has a ribosome binding site that starts the translation of a 3000 amino acid containing protein that is later cut by cellular and viral proteases into 10 active structural and non-structural smaller proteins.


Replication of HCV involves several steps. The viruses need a certain environment to be able to replicate, and must therefore first move to such areas.

HCV has a high rate of replication with approximately one trillion particles produced each day in an infected individual. Due to lack of proofreading by the HCV RNA polymerase, HCV also has an exceptionally high mutation rate, a factor that may help it elude the host's immune response.

HCV mainly replicates within hepatocytes in the liver, although there is controversial evidence for replication in lymphocytes or monocytes. By mechanisms of host tropism, the viruses reach these proper locations. Circulating HCV particles bind to receptors on the surfaces of hepatocytes and subsequently enter the cells. Two putative HCV receptors are CD81 and human scavenger receptor class B1 (SR-BI). However, these receptors are found throughout the body. The identification of hepatocyte-specific cofactors that determine observed HCV liver tropism are currently under investigation.

Once inside the hepatocyte, HCV initiates the lytic cycle. It utilizes the intracellular machinery necessary to accomplish its own replication. Specifically, the HCV genome is translated to produce a single protein of around 3011 amino acids. This "polyprotein" is then proteolytically processed by viral and cellular proteases to produce three structural (virion-associated) and seven nonstructural (NS) proteins. Alternatively, a frameshift may occur in the Core region to produce an Alternate Reading Frame Protein (ARFP). HCV encodes two proteases, the NS2 cysteine autoprotease and the NS3-4A serine protease. The NS proteins then recruit the viral genome into an RNA replication complex, which is associated with rearranged cytoplasmic membranes. RNA replication takes places via the viral RNA-dependent RNA polymerase of NS5B, which produces a negative-strand RNA intermediate. The negative strand RNA then serves as a template for the production of new positive-strand viral genomes. Nascent genomes can then be translated, further replicated, or packaged within new virus particles. New virus particles presumably bud into the secretory pathway and are released at the cell surface.


Based on genetic differences between HCV isolates, the hepatitis C virus species is classified into nine genotypes (1-9) with several subtypes within each genotype (represented by letters). Subtypes are further broken down into quasispecies based on their genetic diversity. The preponderance and distribution of HCV genotypes varies globally. For example, in North America, genotype 1a predominates followed by 1b, 2a, 2b, and 3a. In Europe, genotype 1b is predominant followed by 2a, 2b, 2c, and 3a. Genotypes 4 and 5 are found almost exclusively in Africa. Genotype is clinically important in determining potential response to interferon-based therapy and the required duration of such therapy. Genotypes 1 and 4 are less responsive to interferon-based treatment than are the other genotypes (2, 3, 5 and 6). Duration of standard interferon-based therapy for genotypes 1 and 4 is 48 weeks, whereas treatment for genotypes 2 and 3 is completed in 24 weeks.


Unlike hepatitis A and B, there is currently no vaccine to prevent hepatitis C infection.

In a 2006 study, 60 patients received four different doses of an experimental hepatitis C vaccine. All the patients produced antibodies that the researchers believe could protect them from the virus.

A treatment for Hepatitis C exists which includes the use of PEG (for interferon longevity) and ribavirin (which interferes with HCV reproduction). The treatment, however is primarily palliative.

Cetuximab (Erbitux)

Cetuximab (IMC-C225—marketed under the name Erbitux) is a chimeric (mouse/human) monoclonal antibody, an epidermal growth factor receptor (EGFR) inhibitor, given by intravenous infusion for treatment of metastatic colorectal cancer and head and neck cancer.
 Cetuximab is indicated for the treatment of patients with epidermal growth factor receptor (EGFR)-expressing, KRAS wild-type metastatic colorectal cancer (mCRC), in combination with chemotherapy, and as a single agent in patients who have failed oxaliplatin- and irinotecan-based therapy and who are intolerant to irinotecan. The positive opinion from the Committee for Medicinal Products for Human Use (CHMP) was received for mCRC 1st line use in May 2008

Cetuximab (Erbitux) is indicated for the treatment of patients with squamous cell carcinoma of the head and neck in combination with platinum-based chemotherapy for the 1st line treatment of recurrent and/or metastatic disease and in combination with radiation therapy for locally advanced disease. The positive CHMP opinion for this indication was received in October 2008.

A diagnostic immunohistochemistry assay (EGFR pharmDx) can be used to detect EGFR expression in the tumor material. Approximately 75% of patients with metastatic colorectal cancer have an EGFR-expressing tumor and are therefore considered eligible for treatment with cetuximab or panitumumab 

Potent Dual Inhibitor of JAK 1/2 in Phase I/II Clinical Development