Post-translational modification refers to the covalent and enzymatic modification of proteins following protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may undergo PTM to form the mature protein product. PTMs are important components in cell signaling, as for example when prohormones are converted to hormones. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini, they can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Phosphorylation is a common mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic proteins have carbohydrate molecules attached to them in a process called glycosylation, which can promote protein folding and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation targets a protein or part of a protein attached to the cell membrane.
Other forms of post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may be referred to as a post-translational modification. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, a propeptide is removed from the middle of the chain; some types of post-translational modification are consequences of oxidative stress. Carbonylation is one example that targets the modified protein for degradation and can result in the formation of protein aggregates. Specific amino acid modifications can be used as biomarkers indicating oxidative damage. Sites that undergo post-translational modification are those that have a functional group that can serve as a nucleophile in the reaction: the hydroxyl groups of serine and tyrosine. In addition, although the amide of asparagine is a weak nucleophile, it can serve as an attachment point for glycans.
Rarer modifications can occur at some methylenes in side chains. Post-translational modification of proteins can be experimentally detected by a variety of techniques, including mass spectrometry, Eastern blotting, Western blotting. Additional methods are provided in the external links sections. Myristoylation, attachment of myristate, a C14 saturated acid palmitoylation, attachment of palmitate, a C16 saturated acid isoprenylation or prenylation, the addition of an isoprenoid group farnesylation geranilgeranilatyon glipyatyon, glycosylphosphatidylinositol anchor formation via an amide bond to C-terminal tail lipoylation, attachment of a lipoate functional group flavin moiety may be covalently attached heme C attachment via thioether bonds with cysteines phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, non-ribosomal peptide and leucine biosynthesis retinylidene Schiff base formation diphthamide formation ethanolamine phosphoglycerol attachment hypusine formation beta-Lysine addition on a conserved lysine of the elongation factor P in most bacteria.
EFP is an homolog to eIF5A and aIF5A. Acylation, e.g. O-acylation, N-acylation, S-acylation acetylation, the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues. See histone acetylation; the reverse is called deacetylation. Formylation alkylation, the addition of an alkyl group, e.g. methyl, ethyl methylation the addition of a methyl group at lysine or arginine residues. The reverse is called demethylation. Amidation at C-terminus. Formed by oxidative dissociation of a C-terminal Gly residue. Amide bond formation amino acid addition arginylation, a tRNA-mediation addition polyglutamylation, covalent linkage of glutamic acid residues to the N-terminus of tubulin and some other proteins. Polyglycylation, covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail butyrylation gamma-carboxylation dependent on Vitamin K glycosylation, the addition of a glycosyl group to either arginine, cysteine, serine, tyrosine, or tryptophan resulting in a glycoprotein.
Distinct from glycation, regarded as a nonenzymatic attachment of sugars. Polysialylation, addition of polysialic acid, PSA, to NCAM malonylation hydroxylation: addition of an oxygen atom to the side-chain of a Pro or Lys residue iodination: addition of an iodine atom to the aromatic ring of a tyrosine residue nucleotide addition such as ADP-ribosylation phosphate ester or phosphoramidate formation phosphorylation, the addition of a phosphate group to serine and tyrosine, or histidine adenylylation, the addition of an adenylyl moiety to tyrosine, or histidine and lysine uridylylation, the addition of an uridylyl-group to tyrosine propionylation pyroglutamate formation S-glutathionylation S-nitrosylation S-sulfenylation, reversible covalent
In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers; the word dimer has roots meaning "two parts", di- + -mer. A protein dimer is a type of protein quaternary structure. A protein homodimer is formed by two identical proteins. A protein heterodimer is formed by two different proteins. Most protein dimers in biochemistry are not connected by covalent bonds. An example of a non-covalent heterodimer is the enzyme reverse transcriptase, composed of two different amino acid chains. An exception is dimers that are linked by disulfide bridges such as the homodimeric protein NEMO; some proteins contain specialized domains to ensure specificity. Antibodies Receptor tyrosine kinases Transcription factors Leucine zipper motif proteins Nuclear receptors 14-3-3 proteins G protein-coupled receptors G protein βγ-subunit dimer Kinesin Triosephosphateisomerase Alcohol dehydrogenase Factor XI Factor XIII Toll-like receptor Fibrinogen Variable surface glycoproteins of the Trypanosoma parasite Tubulin Type II restriction enzymes Dimer Protein trimer Oligomer ProtCID
A slit lamp is an instrument consisting of a high-intensity light source that can be focused to shine a thin sheet of light into the eye. It is used in conjunction with a biomicroscope; the lamp facilitates an examination of the anterior segment and posterior segment of the human eye, which includes the eyelid, conjunctiva, natural crystalline lens, cornea. The binocular slit-lamp examination provides a stereoscopic magnified view of the eye structures in detail, enabling anatomical diagnoses to be made for a variety of eye conditions. A second, hand-held lens is used to examine the retina. Two conflicting trends emerged in the development of the slit lamp. One trend originated from clinical research and aimed to apply the complex and advanced technology of the time; the second trend originated from ophthalmologic practice and aimed at technical perfection and a restriction to useful methods. The first man credited with developments in this field was Hermann von Helmholtz when he invented the ophthalmoscope.
In ophthalmology and optometry, the instrument is called a "slit lamp", although it is more called a "slit lamp instrument". Today's instrument is a combination of two separate developments, the corneal microscope and the slit lamp itself; the first concept of a slit lamp dates back to 1911 credited to Allvar Gullstrand and his "large reflection-free ophthalmoscope." The instrument was manufactured by Zeiss and consisted of a special illuminator connected to a small stand base through a vertical adjustable column. The base was able to move on a glass plate; the illuminator employed a Nernst glower, converted into a slit through a simple optical system. However, the instrument never received much attention and the term "slit lamp" did not appear in any literature again until 1914, it wasn't until 1919 that several improvements were made to the Gullstrand slit lamp made by Vogt Henker. First, a mechanical connection was made between ophthalmoscopic lens; this illumination unit was mounted to the table column with a double articulated arm.
The binocular microscope was supported on a small stand and could be moved across the tabletop. A cross slide stage was used for this purpose. Vogt introduced Koehler illumination, the reddish Nernst glower was replaced with the brighter and whiter incandescent lamp. Special mention should be paid to the experiments that followed Henker's improvements in 1919. On his improvements the Nitra lamp was replaced with a carbon arc lamp with a liquid filter. At this time the great importance of color temperature and the luminance of the light source for slit lamp examinations were recognized and the basis created for examinations in red-free light. In the year 1926, the slit lamp instrument was redesigned; the vertical arrangement of the projector made it easy to handle. For the first time, the axis through the patient's eye was fixed along a common swiveling axis, although the instrument still lacked a coordinate cross-slide stage for instrument adjustment; the importance of focal illumination had not yet been recognized.
In 1927, stereo cameras were developed and added to the slit lamp to further its use and application. In 1930, Rudolf Theil further developed the slit lamp, encouraged by Hans Goldmann. Horizontal and vertical co-ordinate adjustments were performed with three control elements on the cross-slide stage; the common swivel axis for microscope and illumination system was connected to the cross-slide stage, which allowed it to be brought to any part of the eye to be examined. A further improvement was made in 1938. A control lever or joystick was used for the first time to allow for horizontal movement. Following World War II the slit lamp was improved again. On this particular improvement the slit projector could be swiveled continuously across the front of the microscope; this was improved again in 1950. They adopted the joystick control from the Goldmann instrument and the illumination path present in the Comberg instrument. Additionally, Littmann added the stereo telescope system with a common objective magnification changer.
In 1965, the Model 100/16 Slit Lamp was produced based on the slit lamp by Littmann. This was soon followed by the Model 125/16 Slit Lamp in 1972; the only difference between the two models was their operating distances of 100 mm to 125 mm. With the introduction of the photo slit lamp further advancements were possible. In 1976, the development of the Model 110 Slit Lamp and the 210/211 Photo Slit Lamps were an innovation by which each were constructed from standard modules allowing for a wide range of different configurations. At the same time, halogen lamps replaced the old illumination systems to make them brighter and daylight quality. From 1994 onwards, new slit lamps were introduced; the last major development was in 1996 in. See "From Lateral Illumination to Slit Lamp - An Outline of Medical History". While a patient is seated in the examination chair, they rest their chin and forehead on a support to steady the head. Using the biomicroscope, the ophthalmologist or optometrist proceeds to examine the patient's eye.
A fine strip of paper, stained with fluorescein, a fluorescent dye, may be touched to the side of the eye. The dye is rinsed out of the eye by tears. A subsequent test may involve placing drops in the eye; the drops take about 15 to 20 minutes to work, after which the examination is repeated, allowing the back of the eye to be examined. Patients will experience some light sensitivity for a few hours after this exam, and
Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteins are tagged for degradation with a small protein called ubiquitin; the tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules; the result is a polyubiquitin chain, bound by the proteasome, allowing it to degrade the tagged protein. The degradation process yields peptides of about seven to eight amino acids long, which can be further degraded into shorter amino acid sequences and used in synthesizing new proteins. Proteasomes are found inside all eukaryotes and archaea, in some bacteria. In eukaryotes, proteasomes are located both in the cytoplasm.
In structure, the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins; the inner two rings are made of seven β subunits. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded; the outer two rings each contain seven α subunits whose function is to maintain a "gate" through which proteins enter the barrel. These α subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process; the overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system. The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, responses to oxidative stress; the importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways was acknowledged in the award of the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose.
Before the discovery of the ubiquitin proteasome system, protein degradation in cells was thought to rely on lysosomes, membrane-bound organelles with acidic and protease-filled interiors that can degrade and recycle exogenous proteins and aged or damaged organelles. However, work by Joseph Etlinger and Alfred Goldberg in 1977 on ATP-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the presence of a second intracellular degradation mechanism; this was shown in 1978 to be composed of several distinct protein chains, a novelty among proteases at the time. Work on modification of histones led to the identification of an unexpected covalent modification of the histone protein by a bond between a lysine side chain of the histone and the C-terminal glycine residue of ubiquitin, a protein that had no known function, it was discovered that a identified protein associated with proteolytic degradation, known as ATP-dependent proteolysis factor 1, was the same protein as ubiquitin.
The proteolytic activities of this system were isolated as a multi-protein complex called the multi-catalytic proteinase complex by Sherwin Wilk and Marion Orlowski. The ATP-dependent proteolytic complex, responsible for ubiquitin-dependent protein degradation was discovered and was called the 26S proteasome. Much of the early work leading up to the discovery of the ubiquitin proteasome system occurred in the late 1970s and early 1980s at the Technion in the laboratory of Avram Hershko, where Aaron Ciechanover worked as a graduate student. Hershko's year-long sabbatical in the laboratory of Irwin Rose at the Fox Chase Cancer Center provided key conceptual insights, though Rose downplayed his role in the discovery; the three shared the 2004 Nobel Prize in Chemistry for their work in discovering this system. Although electron microscopy data revealing the stacked-ring structure of the proteasome became available in the mid-1980s, the first structure of the proteasome core particle was not solved by X-ray crystallography until 1994.
The proteasome subcomponents are referred to by their Svedberg sedimentation coefficient. The proteasome most used in mammals is the cytosolic 26S proteasome, about 2000 kilodaltons in molecular mass containing one 20S protein subunit and two 19S regulatory cap subunits; the core provides an enclosed cavity in which proteins are degraded. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites. An alternative form of regulatory subunit called the 11S particle can associate with the core in the same manner as the 19S particle; the number and diversity of subunits contained in the 20S core particle depends on the organism. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits; the α subunits are pseudoenzymes homologous to β subunits. They are assembled with their N-termini adjacent to
Chromosome 3 is one of the 23 pairs of chromosomes in humans. People have two copies of this chromosome. Chromosome 3 spans 200 million base pairs and represents about 6.5 percent of the total DNA in cells. The following are some of the gene count estimates of human chromosome 3; because researchers use different approaches to genome annotation their predictions of the number of genes on each chromosome varies. Among various projects, the collaborative consensus coding sequence project takes an conservative strategy. So CCDS's gene number prediction represents a lower bound on the total number of human protein-coding genes; the following is a partial list of genes on human chromosome 3. For complete list, see the link in the infobox on the right. Partial list of the genes located on p-arm of human chromosome 3: Partial list of the genes located on q-arm of human chromosome 3: The following diseases and disorders are some of those related to genes on chromosome 3: National Institutes of Health. "Chromosome 3".
Genetics Home Reference. Retrieved 2017-05-06. "Chromosome 3". Human Genome Project Information Archive 1990–2003. Retrieved 2017-05-06
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization; the method is named after the British biologist Edwin Southern, who first published it in 1975. Other blotting methods that employ similar principles, but using RNA or protein, have been named in reference to Edwin Southern's name; as the label is eponymous, Southern is capitalised. The names for other blotting methods may follow this convention, by analogy. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments; the DNA fragments are electrophoresed on an agarose gel to separate them by size. If some of the DNA fragments are larger than 15 kb prior to blotting, the gel may be treated with an acid, such as dilute HCl; this depurinates the DNA fragments, breaking the DNA into smaller pieces, thereby allowing more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for hybridization to the probe, destroys any residual RNA that may still be present in the DNA; the choice of alkaline over neutral transfer methods, however, is empirical and may result in equivalent results. A sheet of nitrocellulose membrane is placed on top of the gel. Pressure is applied evenly to the gel, to ensure good and contact between gel and membrane. If transferring by suction, 20X SSC buffer is used to prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential is used to move the DNA from the gel onto the membrane; the membrane is baked in a vacuum or regular oven at 80 °C for 2 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane.
The membrane is exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, detergents such as SDS to reduce non-specific binding of the probe. After hybridization, excess probe is washed from the membrane, the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of colour on the membrane if a chromogenic detection method is used. Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence, complementary to the probe.
The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods. Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will be observed when the probe hybridizes to several similar sequences. Modification of the hybridization conditions may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar. Southern blotting transfer may be used for homology-based cloning on the basis of amino acid sequence of the protein product of the target gene. Oligonucleotides are designed; the oligonucleotides are chemically synthesized and used to screen a DNA library, or other collections of cloned DNA fragments.
Sequences that hybridize with the hybridization probe are further analysed, for example, to obtain the full length sequence of the targeted gene. Southern blotting can be used to identify methylated sites in particular genes. Useful are the restriction nucleases MspI and HpaII, both of which recognize and cleave within the same sequence. However, HpaII requires that a C within that site be methylated, whereas MspI cleaves only DNA unmethylated at that site. Therefore, any methylated sites within a sequence analyzed with a particular probe will be cleaved by the former, but not the latter, enzyme. Ge
Hydroxylation is a chemical process that introduces a hydroxyl group into an organic compound. In biochemistry, hydroxylation reactions are facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of organic compounds in air, it is important in detoxification since hydroxylation converts lipophilic compounds into water-soluble products that are more removed by the kidneys or liver and excreted. Some drugs are deactivated by hydroxylation; the hydroxylation process involves conversion of a CH group into a COH group. Hydroxylation is an oxidative process; the oxygen, inserted into the C-H bond is derived from atmospheric oxygen. Since O2 itself is a slow and unselective hydroxylating agent, catalysts are required to accelerate the pace of the process and to introduce selectivity; the principal hydroxylation agent in nature is cytochrome P-450, hundreds of variations of which are known. Other hydroxylating agents include flavins, alpha-ketoglutarate-dependent hydroxylases, some diiron hydroxylases.
The most hydroxylated residue in human proteins is proline. This is due to the fact that collagen makes up about 25–35% of the protein in our bodies and contains a hydroxyproline at every 3rd residue in its amino acid sequence. Hydroxylation occurs at the γ-C atom, forming hydroxyproline, which stabilizes the secondary structure of collagen due to the strong electronegative effects of oxygen. Proline hydroxylation is a vital component of hypoxia response via hypoxia inducible factors. In some cases, proline may be hydroxylated instead on its β-C atom. Lysine may be hydroxylated on its δ-C atom, forming hydroxylysine; these three reactions are catalyzed by large, multi-subunit enzymes prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl 5-hydroxylase, respectively. These reactions require iron to carry out the oxidation, use ascorbic acid to return the iron to its reduced state. Deprivation of ascorbate leads to deficiencies in proline hydroxylation, which leads to less stable collagen, which can manifest itself as the disease scurvy.
Since citrus fruits are rich in vitamin C, British sailors were given limes to combat scurvy on long ocean voyages. 17α-Hydroxylase Cholesterol 7 alpha-hydroxylase Dopamine β-hydroxylase Phenylalanine hydroxylase Tyrosine hydroxylase