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TA5
Biotechnology: Bringing peptide science to your skin
In the cosmetic industry biotechnology in synonymous with innovation. Literally speaking the technology of life has emerged
as the conrnerstone of modern cosmetology. The foundation of biotechnology to develop cosmetic ingredients with added value.
While years ago, the cosmetics industry was solely “Market Driver”. It now thrives or “the science locomotive”
fulled by the advent of biotechnologies.
The mission of Biotechnology in Cosmetology
is to constantly innovate. The new pathways being highlighted by biotechnology provide formulation. Chemist with adequat bioactive
tools to enhance their finish products. The main advantage of Biotechnology derived ingredient residue in the specificity
of their action. The most popular and innovative technology in bio-science is peptide synthesis. These active molecules (What is peptide?) will eventually lead to
tailored-made cosmetic products that will work at preventuly against or even alleviating, undesirable skin phenotype. We wish
to make available ingredients that will fine-tune their action according to the targeted tissue, sunlighted intensity stress
level and cellular, metabolic activity. What may seem science fiction today may as well be part of our daily skin care regimen
tomorrow with the help of TA5.
To see more information about Amino
Acids, Peptides, and Proteins: Go Further down
What is TA5?
Recent discoveries have shown that hydralytic fraction of the native collagen strand-a peptide signal sequence-in capable
of stimulating the growth of fiberblasr-like cells and industry collagen synthesis. Modification of this peptide sequence
generated a novel peptide signal sequence, which in turn was complexed to two cofactors known to be integral components in
the collagen synthetic pathway; namely, ascorbic palmitale and electron iron. The complex is named TA5, which is tetra-peptide.
Action of TA5
The standard consequence of exposure to TA5 are best illustrated in Vitro with
the proline incorporation array. Since proline is an integral amino acid of collagen quantification of proline incorporation
may be used to measure potential collagen deposition stimulated by TA5. TA5 consist of four amino acid, one of the amino acid,
which is proline, because hydroxylated and start the production of native collagen.
TA5, a novel peptide signal sequence
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INTRODUCTION
BRIEF
ABSTRACT
By applying the properties
of peptide signal sequences as genetic and metabolic feedback messengers, a novel peptide signal sequence complex was generated
that significantly affects dermal metabolism and structure. Tests using this sequence have demonstrated a dose-dependent increase
in fibroblast proliferation and an enhanced ability of the fibroblast to thrive in a stressful environment. Similar testing
with murine hepatocytes revealed a significant increase in cellular respiration. The morphological changes associated with
these metabolic responses include an increase in proline incorporation in the dermal matrix and an increase in the concentration
of lysyl oxidase. These findings may contribute to accelerated regeneration of the dermal matrix, its enhanced durability,
and an acceleration of the wound healing response.
ABSTRACT
The synthesis of a novel peptide, TA5's signal sequence has been shown in
early testing to significantly reverse some of the in vitro endpoints of cutaneous aging with positive clinical correlation.
The signal sequence described has several independent mechanisms of action that include selective, dose-dependent fibroblast
proliferation, an increase in the metabolic activity of the skin, and increase in collagen I and III syntheses as measured
by a proline incorporation assay (with more than twice the end result when compared to retinoic acid 0025%), depression of
tyrosinase activity, and a down-regulatory effect on proinflammatory prostaglandins in in vitro three-dimensional skin models.
In vivo test results have demonstrated a significant acceleration of the wound healing response with associated anti-erythema
properties. Pilot clinical studies have revealed an average 8% increase in epidermal thickness 6 weeks after application and
a high level of patient satisfaction (p = 0.0225) regarding qualitative cosmetic changes in a population previously treated
with retinoic acid or chemical peels.
SPECTRUM
OF ACTIVITY FOR TA5
TA5 has been shown to selectively upregulate fibroblast proliferation and respiration while
increasing fibroblast durability under unfavorable conditions. The structural consequences of these metabolic changes in the
dermis include increased proline incorporation and accelerated collagen deposition. In addition, the observed elevation in
lysyl oxidase concentrations may contribute to an accelerated maturation of the collagen matrix, as well as to an enhanced
remodeling. phase during the wound healing process.
Clearly, the concept of signal sequence technology and its potential
applications in genetics and medical therapeutics warrants further investigation. The experiments presented in this paper
explored some of the metabolic and morphological consequences of applying a novel signal sequence complex, TA5, to the skin.
The findings presented raise a number of interesting questions and possibilities. In particular, what is the relationship,
if any, between TA5, the observed metabolic changes, and any genetic up - or down-regulation? Secondly, what is the relationship
between proline deposition and changes in cellular metabolism? And thirdly, what is the potential of TA5 to counter the effects
of dermatohetiosis and intrinsic aging of the skin?
Since aging may be defined as the progressive, tirne-dependent
deterioration of an organ system's structural or functional integrity, it is conceptually reasonable to conclude that TA5’s
ability to enhance the dermis' metabolism and structure contributes to a regeneration of the dermis and a reversal of some
of the aging process. The basic science and clinical findings demonstrative of intrinsic aging and dermatohehosis are well
established and have served as the basis for exploring treatment options that reduce or reverse signs of aging using different
topical preparations. The majority of preparations include the familiar retinoic acid derivatives and alphahydroxy and beta-hydroxy
acids. Based on our current understanding of TA5, it should be positioned as a new class of active, with expected clinical
efficacy beyond that of the retinoic acid derivatives.
It is hypothesized that the spectrum of activity for TA5 is
far broader than our current experimentation has revealed. The results obtained in the case study presented are promising
and suggest that TA5 may also exhibit an ability to counter the inflammatory response and minimize scarring potential. Further
studies into wound heating are clearly warranted.
COMMENTS
According to the pilot study conducted
at Ball Memorial Hospital, an affiliate of Indiana-
University
School of Medicine, on women between the ages of 28 and so with moderate sun damage and age-induced brown spots and wrinkles.
TA5 increased skin thickness by an average of 8% and reduced irregular pigmentation
by 54%. Skin was thickened due to increased production of Collagen I and Collagen III which are the key players in minimizing
wrinkles.
Alongside this remarkable result, TA5 treatments were non-irritating to the skin and clinically demonstrated their ability to reduce
wrinkles, facial pigmentation spots, and other signs of aging and sun damage.
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RESEARCHER'S COMMENTS
"The results are very exciting. If you think about Retin-A™ and alpha hydroxy acid as first generation, this is
clearly a second generation product. It works better and is totally non-irritating to the skin."
Brian Donnelson,
principal
investigator of the study and director of the Dermatology Research Laboratory at Ball Memorial Hospital. |
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CLINICAL SUBJECT COMMENTS
"I was really skeptical about TA5’s ability to get rid of wrinkles and make me look younger. But the product really works. I saw
a definite improvement in my skin’s appearance and a real reduction in wrinkles, particularly the crow’s feet
around my eyes. I’ve tried many other products, including Retin-A and alpha hydroxy. TA5 is better and doesn’t irritate the skin at all."
–
Jerilyn Justice |
Comment: Clinical trials of TA5 were conducted
at Ball Memorial Hospital, an affiliate of Indiana University School of Medicine. All clinical participants had previously
used either Retin-A, alpha hydroxy acid, or glycolic acid products, but not within three months prior to the study.
COMPARISON
Researchers state that TA5 is five times
as effective as Retin-A at decreasing lines, wrinkles, and blemishes.
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REPLACEMENT |
TA5™ |
Retin-A™ |
Collagen |
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Side Effects |
none |
photosensivity |
minor to severe
allergic reaction |
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Safe for Eyelids |
yes |
no |
no |
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Pre-test for Sensitivity |
none |
yes |
yes |
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% Proline Incorporation |
360% |
134.2% |
Non Applicable |
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Peptide Technology
Amino Acids, Peptides, and Proteins
The a Amino Acids
The general structure of the amino acids found in proteins could be depicted as:
As their name implies, they all contain a carboxylic acid
group and an amino group. They are called alpha (a) amino acids
based upon an archaic system of nomenclature. In this system the carbon adjacent to a carboxyl carbon is designated the a carbon. Since the naturally occurring amino acids have the amino group on the a carbon they are a amino acids.The
carboxylic acid group is a weak acid with a pKa in the range of 1.7 to 2.6. The carboxylic acid groups of the a amino acids are stronger acids than simple organic acids. The amino group is a weak base having a pKa in the range of 8.8 to 10.8. Since
the carboxyl group is an acid and the amino group is a base, in solution amino acids self neutralize / autoneutralize. The structure given previously for the naturally occurring amino acids is incorrect, the correct structure for an amino acid in solution is:
This DIPOLAR IONIC form exists when the amino acids are
in solution at neutral pH and when they are in the solid state.
The dipolar ionic form explains many of the properties of the amino acids.
For example their melting points, boiling points, and solubilities more closely resemble those of ionic salts rather than organic acids. The dipolar ionic form of an amino acid is called a ZWITTER ION. Any molecular compound that contains a mixture of positive and negative charges is a zwitter ion.
pH Properties of the Amino Acids
The ionic state of the amino acids depends upon the pH
of the solution in which they are dissolved.At low pH (pH ~ 1.0)
the amino acid is in the cationic form and if placed in an electric field they will migrate toward the cathode. As the pH is increased the carboxylic acid group ionizes. When the pH is equal to the pKa the amino acid exists as a 50:50 mixture of the cationic and zwitter ionic forms. Adding more base results in continued ionization of the carboxylic acid group
until the zwitter ionic form is the predominant form of
the amino acid in solution. By the addition of more base, the pKa of the amino group is reached and at this point the amino acid exists as a 50:50 mixture of the zwitter ionic form and the anionic form. As the pH is increased further the amino group
continues loses its proton and ultimately, at high pH (pH ~ 12.0),
the anionic form is the predominant form in solution. The anionic
form will migrate toward the anode if placed in an electric field. This titration curve demonstrates that the a amino acids never exist in an uncharged form. As the amino acid is titrated, there is a solution pH along the titration curve where the amount of positive charge on the molecule is exactly balanced by the amount of negative charge. At this pH the amino acid in solution has no net charge. The amount of positive charge is exactly balanced by an equal amount of negative charge. The solution pH at which the opposite charges exactly balance one another is called the ISOELECTRIC POINT (pI) of the amino acid.
At the isoelectric point an amino acid will not migrate
in an electric field since it has no net charge.At pH’s below the
isoelectric point, the amino acid has a net positive charge and it will migrate toward
the cathode; above the isoelectric point the molecule has a net negative charge and it will migrate
toward the anode.
The R Groups - The Amino Acid Side Chains
The different R groups, the different amino acid
side chains give each of the 20 amino acids different chemical and physical
properties. Different combinations of the amino acids within a protein give the
protein its unique chemical and physical properties. The unique sequence of amino acids defines the
structure and function of the protein.Its time to examine the structure
of the side chains of the 20 naturally occurring amino acids The amino
acids can be divided into two major groups: Nonpolar (Hydrophobic) Amino Acids and Polar
(Hydrophilic) Amino Acids. The Polar Amino Acids can be divided into three subgroups:
Polar But Not Charged Amino Acids, Amino Acids with
Negatively Charged Side Chains (Acidic Amino Acids), and Amino Acids
with Positively Charged Side Chains (Basic Amino Acids).
The Hydrophobic (Nonpolar) Amino Acids are
Note 1: The amino acids Tryptophan (Trp) and Glycine (Gly)
are sometimes included in the polar but not charged group of amino
acids. Tryptophan is sometimes included in this group because the nitrogen
in the ring system makes it slightly polar. However, the complex ring system is fairly nonpolar and overall Trp behaves most often like a nonpolar amino acid. Glycine is included because its side chain is the small H and because its solubility in water resembles that of the other polar amino acids.
The Polar But Not Charged Amino Acids are:
Note 2: The side chains of cysteine and tyrosine have some
acid / base properties. The -SH group on cysteine has a pKa of
8.3 and the -OH group on the tyrosine side chain has a pKa of 10.1. These pKa’s can be measured in vitro (in a test tube). At physiological pH, pH 7.4, these side chains are for
the most part unionized.
Note 3: Phenylalanine, Tyrosine and Tryptophan are called
the aromatic amino acids because they all contain an aromatic
ring as part of their side chains. The aromatic groups on the side chains of Phenylalanine, Tyrosine and Tryptophan absorb light at the ultraviolet region of the spectrum, at 280 nm. This property is employed by biochemists to detect and quantify proteins in solutions and / or biological fluids.
The Amino Acids with Negatively Charged Side Chains (Acidic
Amino Acids) are:
The side chain carboxyl group of aspartate (aspartic acid)
has a pKa of 3.9 and the side chain carboxyl group of glutamate
(glutamic acid) has a pKa of 4.3. These values are closer the pKa’s of simple organic acids.
The Basic Amino Acids are:
The imidazole ring of histidine has a pKa of 6.0, the side
chain amino group of lysine has a pKa of 10.5, and the guanidinium
group of arginine has a pKa of 12.5.
Chiral Carbon
When the R group (side chain) of an amino acid is an organic
group other than a hydrogen (H) atom, the a carbon of the molecule
is an asymmetric carbon; a chiral carbon (a tetrahedral stereocenter).
A chiral carbon in the molecule means that the molecule can exists as a pair of stereoisomers. These stereoisomers have mirror image configurations; they exist as a pair of enantiomers. The pair of enantiomers for an a amino acid have the following configurations:
In nature, when two enantiomers exist, usually only one
is synthesized and used by cells. In the case of the amino acids,
only the L configuration exists in nature and is used by cells. Some bacteria and fungi synthesize and use D configuration amino acids. The molecules synthesized with these D-form amino acids are often toxic to other living organisms. With these few minor exceptions all of the naturally occurring amino acids are in the L-form.
Calculation of Amino Acid Isoelectric Point
For the nonpolar and polar amino acids with two pKa’s,
the isoelectric point is calculated by taking the numerical average
of the carboxyl group pKa and the a-amino group pKa.The titration
curves for seven of the amino acids (Cys, Try, Glu, Asp, His, Lys, & Arg) demonstrate three inflection points, three pKa’s. One for the a-amino group, one for the carboxyl group (carbon
1), and one for the ionizable side chain. The isoelectric
point for these amino acids in calculated by taking the numerical
average of the pKa’s of the groups with like charge when ionized. For example to calculate the isoelectric point of Glu, the pKa’s of the two carboxyl groups are averaged. To calculate the isoelectric point of Arg, the pKa’s of the a-amino group
and the guanidinium group are averaged. Remember, at the isoelectric
point the amino acid has no net charge. For the amino acids with
three ionizable groups, the total charge on the groups with like charge must equal one (1) so that it can be balanced by the one (1) opposite charge present on the molecule.
Proteins: Their Primary Structure and Biological Functions
The word protein comes from the Greek PROTEIOS which means
first or primary. Proteins are of primary importance to cells
and organisms. Proteins can be compared to words of the English language.
The words that are read, written, and spoken are composed of the 26 letters of the alphabet. The sequence of letters is the primary structure of the word. From the 26 letters of the alphabet an infinite number of "words" can be made. Some of these words have meaning, others are just gibberish. For proteins the alphabet is the 20 amino acids. The equence of amino acids is the primary structure of the polypeptide. From these 20 amino acids an infinite number of polypeptides can be made, each with a different primary structure. Many of these polypeptides are just gibberish. The cell only synthesizes proteins that have meaning.
Peptide Bonds
Amino acids are linked in proteins in a head to tail manner
by a condensation reaction between the carboxyl group of one amino
acid and the a amino group of the second. The "back bone" of a protein
consists of the repeating sequence -N-Ca-C-. The amino acid side chains of the amino acids project perpendicularly from the back bone of the molecule. The
bond between amino acids in a protein is an amide bond. Since this amide bond holds peptides and proteins together it is called a PEPTIDE BOND. This amide linkage, the peptide bond, has no acid or base properties. It will neither donate nor accept a proton. However, the peptide bond is very polar.
Physical chemical studies have shown that the peptide bond
exists in two resonance forms:
The real nature of the peptide bond lies somewhere between
these two extremes, it has partial double bond character. The
partial double bond character of the peptide bond restricts free rotation about this bond limiting the possible number of conformations that the peptide or protein can assume. It also places the six atoms of the peptide bond in the same plane.
Peptide / Protein Terminology
The unique sequence of amino acids in a peptide or protein
is termed the Primary (1°) Structure of the Protein. A gene contains
the information necessary for the synthesis of a protein, for the assembly
of the primary structure. The primary structure contains the nformation necessary for the protein to fold into its final three dimensional conformation and once correctly folded to assume its cellular function. If the 1° structure of a protein is changed very often the
final shape changes resulting in a nonfunctional polypeptide.
A molecule containing two amino acids joined by a peptide
bond is a Dipeptide; one with three amino acids held together
by two peptide bonds is a Tripeptide; four is a Tetrapeptide; etc. In general, Peptides contain 12 or fewer amino acid residues. An Oligopeptide contains between 12 and 20 amino acids and a Polypeptide contains greater than 20 amino acids. Twenty appears to be a magic number with respect to peptide/protein structure. Oligopeptides with 20 or fewer amino acids do not fold into, do assume a single low energy conformation, rather they exist in numerous random shapes. Molecules with greater than 20 amino acids very often fold into a single stable conformation, a single low energy conformation. The terms polypeptide and protein are often used interchangeably. However, the term protein is often reserved for molecules that perform some cellular function.
Monomeric Proteins contain a single polypeptide chain.
Some proteins are supramolecular complexes composed of more than
one polypeptide chain. These proteins are called Multimeric Proteins.
Homomultimeric Proteins are composed of several polypeptides all with the same sequence of amino acids, all with the same primary structure. Heteromultimeric Proteins are composed of several different polypeptides; polypeptides with different primary structures.
Sequence Convention
A protein is a long linear sequence of amino acids joined
by peptide bonds. One end of the molecule has a free amino group,
an amino group not involved in a peptide bond. This is the Amino Terminus
or N-Terminus of the protein. The opposite end of the molecule has a free carboxyl group, a carboxyl group not involved in a peptide bond. This end is the Carboxy Terminus, Carboxyl Terminus or C-Terminus of the protein. When biochemists
write the sequence of a protein they use either the three letter or the one letter abbreviation for the amino acids, usually separated by hyphens. The amino terminus is always the Left most amino acid and the carboxyl terminus is always the Right most amino acid.
Biological Functions of Proteins
Within the cell / organism proteins serve a wide range
of important biological functions. Enzymes are the biological
catalysts of the chemical reactions that occur within the cell. Enzymes will be examined in detail shortly. Regulatory Proteins regulate
the activities of the cell and the ability of other proteins to carry out their cellular function. The peptide and protein hormones are regulatory proteins. They play a role in regulating overall metabolism, growth, development, and maintenance of the
organism. Allosteric enzymes can be considered regulatory proteins
since they control key cellular reactions. Gene inducers and gene
repressors are also considered regulatory proteins. Gene inducers stimulate gene expression, they turn genes "on". Gene repressors inhibit gene expression, they turn genes "off". Transport Proteins carry specific substances from one place to another. Membrane transporters carry polar molecules across cell membranes. Hemoglobin transports oxygen from the lungs to the tissues; serum albumin carries a wide variety of drugs and metabolites through out the body.
Storage Proteins provide a reservoir of an essential nutrient.
Myoglobin stores oxygen in skeletal muscle tissue; ferritin stores
iron in the liver and bone marrow; the protein ovalbumin serves as a storage depot of amino acids for developing birds; and casein, the major protein in mammalian milk stores amino acids for growing infants. Structural proteins
provide strength, support, and form to cells, tissues, and organisms. Collagen is the major protein of bone, tendons, and cartilage. a-Keratin is the protein of hair, horns, hooves, and fingernails. There are cytoskeletal protein fibers adjacent to the cell membrane that give the cell shape and support. Tubulin, actin, and spectrin are some of the proteins that
make-up the cytoskeleton.
Contractile and Motile Proteins provide the cell / organism
with motion. Examples include actin and myosin of muscle cells
and tubulin of cilia and flagella. The proteins dynein and kinesin drive the movement of vesicles and organelles along cytoskeletal tracks within the cell. A single protein can serve several functions within a cell. For example actin and tubulin can be classified as structural proteins or motile proteins depending upon their location within the cell.
Scaffold Proteins act as bridges by binding to and localizing
specific proteins to specific sites within the cell. They act
as a site upon which supramolecular complexes are formed. Protective
Proteins play an active role in cell defense or in defense of the organism. Antibodies of the immune system protect the organism from foreign invaders. The hemostasis cascade of proteins protects the organism from excessive blood loss. Toxins, such as snake venoms, also fall into this class of proteins. Exotic Proteins display functions that
do not fit the other classifications. One example is the glue protein
secreted by mussels. This protein allows the mussels to anchor to hard surfaces.
Conjugated Proteins
Conjugated proteins contain nonprotein components integral
to their structure and necessary for their function. The nonprotein
part is called a Prosthetic Group. Conjugated proteins include:
Glycoproteins - the prosthetic groups are carbohydrates.
Lipoproteins - the prosthetic groups are lipids.
Nucleoproteins - the prosthetic groups are nucleotides
or nucleic acids.
Phosphoproteins - have phosphoryl (phosphate) groups in
ester linkage to hydroxyl groups on the protein.
Metalloproteins - contain metal ions attached either by
ionic interactions or by coordinate covalent bonds.
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