Protein Structure & Modifications - Cell Biology Lecture Notes PDF

18 Protein structure & modifications: ILOs By the end of this lecture, students will be able to 1. Describe the different orders of protein structure 2. Deduce importance of protein folding 3. Correlate protein structure to its function 4. Classify biologically import...

18 Protein structure & modifications: ILOs By the end of this lecture, students will be able to 1. Describe the different orders of protein structure 2. Deduce importance of protein folding 3. Correlate protein structure to its function 4. Classify biologically important proteins according to their structure and function 5. Interpret the effect of denaturation on proteins’ structure and function ❖ Orders of protein structure: o Primary Structure of Proteins - The primary structure of a protein is defined by “The linear sequences of amino acid residues linked to each other by peptide bonds”. Protein contain between 50 and 2000 amino acid residues. The amino acid composition of a peptide chain has a profound effect on the physical and chemical properties of proteins. - The amino acids sequences are read from N-terminal (amino acid number 1) to C-terminal ends of the peptide. The primary structure of proteins determines the secondary and tertiary structures which are essential for protein functions. - Many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal proteins are known, this information may be used to diagnose or study the disease. o Secondary Structure of proteins - The next level of structure is the secondary structure of a protein which is “The regular, recurring arrangements of adjacent amino acid residues in a polypeptide chain”. It includes coiling, folding or bending of the polypeptide chain leading to specific structure which is kept by Hydrogen bonds. There are two main types of secondary structure, the ∝ - helix and the β- pleated sheet. Both structures are held in shape by hydrogen bonds which are formed between the carbonyl O of one amino acid and the amino H of another. Both types could co- exist in the same protein. ▪ The α – helix: It is a rigid, spiral structure, consisting of a tightly packed, coiled Page 1 of 5  polypeptide backbone core, with the side chains of the amino acids extending outward from the central axis to avoid interfering with each other. ▪ β-Sheet: Another form of secondary structure in which two or more segments of a polypeptide chain line up next to each other, forming a sheet-like structure held together by hydrogen bonds. o Tertiary Structure of proteins - The three dimensional, folded and biologically active conformation of a protein is referred to as tertiary structure. The structure reflects the overall shape of the molecule. - This structure is stabilized by interactions between side chains, ionic interactions, disulfide bonds, and hydrogen bonds. - “Tertiary” refers to both the folding of domains and to the final arrangement of domains in the polypeptide. But what are the “Domains”? - Domains are the three-dimensional structural part of protein that can fold, function and exist independently of the rest of the protein chain. - Therefore, each domain has special characteristics that are structurally independent of the other domains in the polypeptide chain. - Some proteins only contain a single domain, others may have several domains. Some domains have a clearly defined function, like the coenzyme-binding domain. Other domains, are there probably just for their stability. o Quaternary Structure of proteins - Many proteins are made up of a single polypeptide chain and have only three levels of structure discussed above. However, some proteins are made up of multiple polypeptide chains, also known as subunits. - The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by non-covalent interactions (as hydrogen bonds). They may either function independently of each other or may work cooperatively, as in hemoglobin which contains 4 chains, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen Clinical Implications: Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell. However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as aged individuals. Deposits of misfolded proteins are associated with a number of diseases, most important are Parkinson disease and Alzheimer disease. Page 2 of 5  - Chaperones are a family of proteins that guide proteins along the proper pathways for folding. They protect them when they are in the process of folding, shielding them from anything that might bind and hinder the process. Many chaperone proteins are termed "heat shock" proteins because they are made in large amounts when cells are exposed to heat. Heat, in general, destabilizes proteins and makes misfolding more common. So when it gets really hot, cells need some extra help with their proteins. ❖ What is protein “Denaturation”? Proteins have finite lifetimes. Denaturation involves the destruction of the higher level structural organization of protein in other words it is loss of secondary and tertiary structures with the retention of the primary structure. This occurs due to rupture of the non-covalent bonds while peptide bonds responsible for the primary structure are retained since denaturation reactions are not strong enough to break the peptide bonds. A denatured protein loses its native biological properties since the bonds that stabilize the protein are broken down. Thus the polypeptide chain unfolds itself and remains in the unfolded state. Causes of Denaturation: 1. Physical factors as temperature (above 70 oC), vigorous vibration and ionizing radiation, X- rays and high pressure. 2. Chemical factors as strong acids and alkalis (extremes of PH) also urea. Effects of denaturation: Biological changes: - Loss of biological activity of enzymes and protein hormones. - Changes of antigenic property of proteins. - Denatured proteins are easily digested due to unfolding of the peptide chains. Page 3 of 5  A good example for this is using strong chemicals (perms and relaxing treatments) to change the curly hair to straight, this is done by “denaturation” of the disulfide bonds in the hair that are responsible for it’s the curly appearance, leading to permanent loss of the curly texture. On the other hand using medium heat cannot break down these bonds, it’s only capable of breaking the weak hydrogen bonds which can be easily affected by heat, water and humidity. ❖ How are proteins Classified? There are many classifications for proteins most important are: ▪ Classification based on chemical composition - Simple proteins: they are made up of only amino acids. Examples are: plasma albumin the important transport protein in the blood and collagen the major component of connective tissues that make up tendons, ligaments, skin, and muscles. - Conjugated proteins : they contain in their structure a non-protein portion. Example: Glycoproteins: They are proteins that covalently bind one or more carbohydrate units to the polypeptide backbone. They play an important role in cell signaling, cell attachment and regulating the immune system. Lipoproteins: These are combinations of proteins with lipids. Plasma lipoproteins play a key role in the absorption and transport of lipids. ▪ Classification based on shape o Fibrous proteins - Fibrous proteins are elongated strand-like structures and are usually present in the form of rods or wires. They have only primary and secondary structures. - Fibrous proteins are highly resistant to digestion by enzymes, these proteins are insoluble in water, they have primarily mechanical and structural functions providing external protection, support and shape; in fact, they ensure flexibility and/or strength. - Examples include Keratins which forms nails, hair and a large part of the outer layer of the skin. The different stiffness and flexibility of these structures is a consequence of the number of disulfide bonds that contribute, together with other binding forces, to stabilize the protein structure. And this is the reason why keratins in different tissues give different degrees of flexibility as that in hair vs nails or as different hair types. o Globular proteins - Most of the proteins belong to this class. They have a compact and more or less spherical structure, more complex than fibrous proteins. They are generally soluble in water. - Globular proteins are made up of not only primary, secondary but also tertiary and quaternary structures - Unlike fibrous proteins, they act as: enzymes, hormones, membrane transporters and receptors. Examples of globular proteins are: Myoglobin: It is an oxygen carrier in muscle cells providing oxygen to the working muscle and is responsible for the color of the muscle Page 4 of 5  Hemoglobin: the protein responsible for transferring oxygen from the lungs to the tissues. ▪ Classification based on biological Value This classification provides the ability to identify proteins that provide the greatest benefit for consumption in human’s diet specially for individuals with special dietary needs as athletes, aged individuals or those with chronic diseases Proteins of high biological value (HBV): which contain all the essential amino acids e.g. meat, poultry, fish and dairy products. Proteins of low biological value (LBV): which are proteins deficient in one or more essential amino acid as plant based proteins including legumes and vegetables. Page 5 of 5  19 GENETIC VARIATIONS AND MUTATIONS ILOs By the end of this lecture, students will be able to 1. Recognize the different types of mutation. 2. Predict the effect of types of mutation on amino acid sequence and protein structure. 3. Correlate the phenotypic outcome with type of mutation What is meant by mutations? Mutations are permanent changes in a DNA sequence. This altered DNA sequence can be reflected by changes in the base sequence of mRNA, and, sometimes, by changes in the amino acid sequence of a protein. Mutations can cause genetic diseases. Types of mutation I- Point mutation (single base substitution): (Figure 1) This entails the substitution of the original base in the gene by another. This can take either of two forms: 1- Transition: In which one purine is replaced by another purine or one pyrimidine is replaced by another pyrimidine. e.g. A >>>G or C>>>T 2- Transversion: In which a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine. e.g. T>>>> A, C >>>> A, T>>>>> G, C>>>> G Figure 1. point mutation Single-base substitutions may have no physiologic effect if they occur in a DNA region that is not part of the coding or regulatory regions of a gene. Mutations may alter regulatory sequences, eg, in promoter or enhancer regions, which can affect gene expression. Single-base changes that occur within a coding region of a gene may produce disease alleles Consequences of point mutation (Figure 2) 1- Silent mutation: The codon containing the changed base may code for the same amino acid. 1  For example, if the serine codon UCA is given a different third base (to become, say, UCU), it still codes for serine. This usually happens if the mutation happens in the 3 rd codon.(refer to wobble’s theory in translation lecture) Therefore, this is termed a “silent” mutation without any effect on protein structure. 2. Missense mutation: (Figure 2) The codon containing the changed base may code for a different amino acid. The substitution of an incorrect amino acid may result in variable effects on protein structure (e.g. hemoglobin β- chain). This type of mutation can result in one of the following: a- Acceptable missense mutation AAA>>>>>>>>> AAU (codons) Lysine>>>>>>>> Asparagine (amino acid) 61 This produces apparently normal functional hemoglobin. b- Partially acceptable missense mutation GAA>>>>>>>>>> GUA (codons) Figure 2. Consequences of point mutation Glutamic acid>>> Valine (amino acid) 6 This produces Hb S; it can bind and release O2 although abnormal, but doesn’t function in low O2 saturation. C- Unacceptable missense mutation: CAU>>>>>>>> UAU (codons) Histidine >>>>> Tyrosine (amino acid) 58 This produces Hb M; it cannot transport O2 and the only treatment is repeated blood transfusions. Clinical implications Hereditary hemochromatosis (HH) is one of the most common genetic diseases. It is associated with two well-known missense mutations in the HFE gene (Human homeostatic iron regulator protein). These mutations are used to screen ‘‘at-risk populations’’ for this disorder of iron metabolism, which results in liver damage (cirrhosis), diabetes, skin pigmentation, and heart failure. (refer to cardiovascular module) 2 3) Non-sense mutations The codon containing the changed base may become a termination codon. For example, if the Serine codon UCA is changed at the second base and becomes UAA, the new codon causes premature termination of translation at that point and the production of a shortened (truncated) protein II) Frame shift mutation: (Figure3) It results from deletion (removal) or insertion (addition) of one or more nucleotides in DNA that generates altered m RNAs with different effects on protein structure. The insertion or deletion results in shifting in the way the codons are read, the thing that produces totally different aminoacids. Figure 3. Frameshift mutations III) Trinucleotide repeat expansion: (Figure 4) Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, expansion of the CAG codon in exon 1 of the gene for Huntington protein leads to the insertion of many extra glutamine residues in the protein, causing the neurodegenerative disorder Huntington disease. The additional glutamines result in an abnormally long protein that is cleaved, producing toxic fragments that aggregate in neurons. 3  Also, Fragile X syndrome, the most common cause of mental disability in males results from a similar mechanism Figure 4.Trinucleotide repeat expansion IV). Splice site mutations: Mutations at splice sites can alter the way in which introns are removed from pre-mRNA molecules, producing abnormal proteins. Gene silencing can result from splicing alterations leading to lack of protein production. Polymorphisms A polymorphism is a change in genotype that can result in no change in phenotype or a change in phenotype that is harmless, causes increased susceptibility to a disease, or, rarely, causes the disease. It is traditionally defined as a sequence variation at a given locus (allele) in >1% of a population. Polymorphisms primarily occur in the 98% of the genome that does not encode proteins (that is, in introns and intergenic regions). Types (Figure 4) 1. Single-base changes: About 90% of human genome variation comes in the form of single nucleotide polymorphisms (SNPs, pronounced “snips”), that is, variations that involve just one base. 2. Tandem repeats: Polymorphisms in chromosomal DNA can also arise from the presence of a variable number of tandem repeats (VNTR). These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person but is unique for any given individual and, therefore, serves as a molecular “fingerprint.” Clinical implications  Paternity tests (to prove the parenthood of a father to a baby) rely on fingerprinting (VNTR)  Polymorphisms can increase the susceptibility to diseases such as cancer and cardiovascular diseases.  Polymorphisms can be used for screening people at high risk of certain diseases 4 Figure 4. Types of polymorphism 5  20 REGULATION OF GENE EXPRESSION & ITS MODIFICATION ILOs By the end of this lecture, students will be able to 1. Deduce different mechanisms of regulation of gene expression 2. Correlate how intracellular signaling molecules affect gene expression 3. Outline how drugs can be used to modify gene expression & nuclear signaling What is meant by regulation of gene expression? It is the control on the amount of protein that is being expressed (transcription and translation) from the genetic DNA. Types of gene expression 1- Constitutive gene expression: It is unvarying expression of a gene (i.e. expressed at all times). It is responsible for expression of House Keeping genes, which are needed to maintain viability of cell (e.g. genes expressing glycolysis enzymes). 2- Regulated gene expression: It is the expression of genes whose products (proteins) level changes in response to molecular signals (for example consumption of nutrients, stress, infection…etc).They can be inducible genes or repressible genes. ▪ Inducible genes: They are the genes whose products increase in concentration under particular molecular circumstances, i.e.; positive regulation. ▪ Repressible genes: They are the genes whose products decrease in concentration in response to molecular signals. Regulation of gene expression If you remember the sequence of central dogma of life, you can deduce that regulation of gene expression can occur at different levels: I) At the level of chromatin (Chromatin remodeling) ▪ In eukaryotes, DNA is found complexed with histone and nonhistone proteins to form chromatin. Transcriptionally active, decondensed chromatin (euchromatin) differs from the more condensed, inactive form (heterochromatin) in a number of ways: 1- Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by reversible acetylation, or phosphorylation. Such modifications increase the negative charge of histones, thereby decreasing the strength of their association with negatively charged DNA. This relaxes the nucleosome, allowing transcription factors access to specific regions on the DNA and hence more protein expression. The opposite is true. Page 1 of 5 2-Methylation can occur on cytosine bases in CG-rich regions (CpG islands) in the promoter region of many genes. Transcriptionally active genes are less methylated (hypomethylated) than their inactive counterparts, suggesting that DNA hypermethylation silences gene expression. Clinical implication Some drug can affect gene expression by inducing DNA methylation or histone modification as the use of histone deacetylase inhibitors (HDACi); to kill cancer cells by inducing cell cycle II) At the level of DNA (genes) 1) Gene copy number: ▪ An increase or decrease in the number of copies of a gene can affect the amount of gene product produced. ▪ An increase in copy number (gene amplification) can lead to increased gene expression. An example of this is amplification of the gene coding for the enzyme dihydrofolate reductase (DHFR) (required for the synthesis of thymidine triphosphate (TTP), which is essential for DNA synthesis), leading to increased production of the enzyme. ▪ Another example is gene deletion, with the famous example of RBCs. During development of RBCs, immature erythroblasts contain nuclei that produce mRNA for synthesis of the globin chain of hemoglobin. As the cells maturate, the nuclei are extruded, so that the fully mature red blood cells have no genes, so they can no longer produce mRNA and proteins. Clinical implication Gene amplification is the mechanism by which many tumour cells develop resistance to anticancer drugs. E.g., The anticancer drug, methotrexate, acts by inhibiting the enzyme dihydrofolate reductase. Amplification of DHFR genes by cancer cells makes them less responsive (resistant) to methotrexate. 2. Re-arrangement of DNA: A single gene of immunoglobulins (Antibodies) can produce from 10 9−1011 different immunoglobulins, providing the diversity needed for the recognition of an enormous number of antigens. This diversity is due to the process of DNA rearrangement. The chains of Igs contain segments called constant (C ), variable (V), diversity (D), and joining (J). Each time an IG is produced, re-arrangement of different segments (Except the constant segment) occur to produce a different Ig. (Figure 1) Figure 1.Gene re-arrangement to produce different Igs Page 2 of 5 3. Mobile DNA elements (Transposons (Tn): These are mobile segments of DNA that move in a random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself. Transposition has contributed to structural variation in the genome and the potential to alter gene expression and even to cause disease. III) At the level of transcription For transcription factors to work, they have to interact in harmony with DNA sequences acting as regulatory regions as follows: 1- Basal expression elements: (Figure 2) It consists of the proximal element of TATA box that direct RNA polymerase II to the correct start site(+1) and the upstream element e.g. CAAT box or GC box that specify the frequency of initiation. Figure 2.Basal expression elements 2-Regulated expression elements: (Cis-acting elements): They are specific DNA sequences that are present on the same gene, so termed Cis-elements, and are responsible for regulation of expression. They can exert their effect on transcription even when separated by thousands of base pairs from a promoter. They include Enhancers and Silencers (Refer to gene expression 1: transcription lecture Regulatory proteins Activators and inhibitors (called trans- factors as they are produced by other genes than this being transcribed (Refer to gene expression 1: transcription lecture )(Figure 3) Figure 3.Interaction between basal and regulated expression elements Page 3 of 5 3- Response elements They are sequences on DNA to which signaling molecules bind, causing change in gene expression. a. Signaling molecules binding to intracellular receptors Members of the nuclear receptor superfamily include the steroid hormone (glucocorticoids, mineralocorticoids, androgens, and estrogens), vitamin D, retinoic acid, and thyroid hormone receptors. These molecules, in addition to some metals such as iron, have intracellular receptors (either cytoplasmic or nuclear) When such molecules diffuse inside the cell, they bind to their receptors. The receptor-ligand complex translocates to DNA to bind to its specific response element, which is a pre-defined sequence on DNA. (for e.g hormone response element (HRE), or metal response element). This binding can alter rate of expression of certain genes (for example, binding of steroid to steroid response element, causes increased expression of gluconeogenesis enzymes, binding of vitamin D to its response element causes increased expression of calcium binding protein) b. Signaling molecules binding to cell surface receptors These receptors include those for insulin, epinephrine, and glucagon. This extracellular signal is then transduced to intracellular 2nd messengers to end by phosphorylation and activation of different kinases. One of these is cAMP response element–binding [CREB] protein that can bind to a specific responsive element sequence on DNA and result in transcription of some target genes of metabolism or of growth. Clinical implications: Pharmacological modulation of certain gene transcription is a common mechanism of action of many classes of drugs either: A) Directly by targeting nuclear receptor superfamily (that are ligand activated transcriptional factors), where the drug receptor complex, itself induces activation or suppression of gene transcription. For e.g., all steroid hormones, as Glucocorticoids or related steroid drugs; dexamethasone, are used in treatment of many inflammatory and autoimmune disorders by suppressing the transcription of inflammatory mediators and cytokines. B) Indirectly by targeting cell surface receptors to affect their downstream signalling cascades to finally affect their kinases involved in activation e or inhibit of transcriptional factors. For e.g., Insulin (targeting tyrosine kinase receptors) used in treatment of diabetes mellitus by affecting the genes involved in metabolism. C) Indirectly by interacting with enzymes that activate or inhibit transcriptional factors. For e.g., Calcineurin inhibitors, cyclosporine, inhibit calcineurin to inhibit transcription factors involved in transcription of a cytokine mediator (IL-2) so can be used to suppress increased immune activity during treatment of many autoimmune disorders and in graft rejection. (Refer to cytokines lecture) Page 4 of 5 III-Post-Transcriptional Regulation: Regulation can occur during processing of the primary transcript (hn RNA) and during the transport of mRNA from nucleus to the cytoplasm. 1- Alternative splicing and polyadenylation sites In certain cases, the use of alternative splicing and polyadenylation sites causes different proteins to be produced from the same gene. For example, in parafollicular cells of the thyroid gland, the calcitonin gene produces mRNA that codes for calcitonin. In the brain, the transcript of this gene undergoes alternative splicing and polyadenylation to produce a different protein called calcitonin gene-related protein (CGRP). 2-RNA editing It is change in a single nucleotide on mRNA after it has been transcribed. The nucleotide is changed either by insertion, deletion or substitution. An example of RNA editing occurs in the production of β apoprotein (apoβ) that is synthesized in liver and intestinal cells and serves as a component of the lipoproteins (carriers of lipid in circulation). Although these apoproteins are encoded by the same gene, the version of the protein made in the liver (B-100) contains 4563 amino acid residues, while the (B-48) made in intestinal cells has only 2152 amino acid. This is due to RNA editing in intestinal cells results in a change of a single nucleotide causing the creation of a stop codon earlier than usual and hence a shorter protein. 3-Stability of mRNA  Developmental or environmental stimuli like nutrient levels, cytokines, hormones and temperature shifts as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury affect stability of mRNA and hence amount of protein produced  In addition, a group of RNAs called microRNA act as post-transcriptional regulators of their mRNA causing mRNA degradation and/or translational repression. IV- Regulation at the level of Translation: Most eukaryotic translational controls affect the initiation of protein synthesis. The initiation factors for translation, particularly eukaryotic initiation factor 2 (eIF2), are the focus of these regulatory mechanisms. The action of eIF2 can be inhibited by phosphorylation. V- Post-Translational Regulation: After proteins are synthesized, their lifespan is regulated by proteolytic degradation. Proteins have different half-lives, some last for hours or days, others last for months or years. Some proteins are degraded by lysosomal enzymes, other proteins are degraded by proteases in the cytoplasm. Clinical Implications: Post translationally, many drugs can affect certain protein level. For e.g., Protease inhibitors; antivirals that inhibit post-translational processing of precursor viral proteins and is used in treatment of HIV and Hepatitis C infection. Page 5 of 5  21 Autophagy, Lysosomes, Peroxisomes & cell inclusions ILOs By the end of this lecture, students will be able to 1. Explain the role of autophagy as a cellular sink 2. Describe the origin of lysosomal enzymes and their function in health and disease. 3. Predict the role especially of autolysosome and phagolysosome in physiological conditions 4. Connect the structure of proteasome to its degrative function. 5. Predict the role of peroxisomes in cell adaptation to patterns of stress. 6. Correlate the types of cytoplasmic inclusions to patterns of cell activity 7. Justify the impact of its derangement on cellular health. 1. Lysosomes Lysosomes are membrane bounded cell organelles that have an acidic pH and contain hydrolytic enzymes. It contains at least 40 different types of acid hydrolases, such as sulfatases, proteases, nucleases, lipases that are active in acidic pH. These enzymes are manufactured in the same steps of protein synthesis following the same steps in rER, packed in Golgi complex and released in vesicles from trans Golgi network. Lysosomes receive contents to be digested from late endosomes. Lysosomes aid in digesting phagocytosed microorganisms, cellular debris, and cells but also excess or senescent organelles, such as mitochondria and RER. The various enzymes digest the engulfed material into small, soluble end products that are transported by carrier proteins in the lysosomal membrane from the lysosomes into the cytosol andare either reused by the cell or exported from the cell into the extracellular space. Transport of Substances into Lysosomes Substances destined for degradation within lysosomes reach these organelles in one of three ways: through phagosomes, pinocytotic vesicles, or autophagosomes. 1- Phagosomes: Phagocytosed material, contained within phagosomes, moves toward the interior of the cell. The phagosome joins either a lysosome or a late endosome. The hydrolytic enzymes digest most of the contents of the phagosome, especially the protein and carbohydrate components. Lipids, however, are more resistant to complete digestion, and they remain enclosed within the spent lysosome, now referred to as a residual body. (Fig. 1) 1  Fg 1. Pathways of intracellular digestion by lysosomes 2- Autophagy: The term autophagy is derived from the Greek word meaning 'self- devouring'. Senescent organelles such as mitochondria or the RER, need to be degraded. The organelles in question become surrounded by elements of the endoplasmic reticulum and are enclosed in vesicles called autophagosomes. Fate of autophagosomes: These structures fuse either with late endosomes or with lysosomes and share the same subsequent fate as the phagosome. (Fig. 1) Autophagy is a self-digesting mechanism responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosome. Significance of autophagy Regulation of diverse cellular functions including growth, differentiation, response to nutrient deficit and oxidative stress, cell death, and macromolecule and organelle turnover. Mechanism Autophagosome formation is regulated by dozens of “autophagy-related genes” called Atgs. Mutation leads to formation of a double-membrane vesicle, which encapsulates cytoplasm, malformed proteins, long-lived proteins, and organelles and then fuses with lysosomes for degradation. 2  Autophagy Regulation Autophagy is activated in response to diverse stress and physiological conditions. For example, food deprivation, hyperthermia, and hypoxia, which are known as major environmental modulators of ageing, are also conditions that induce autophagy. Figure 2 - Stages of autophagy Autophagy and Diseases Autophagy is important in normal development and responds to changing environmental stimuli. On starvation, autophagy is greatly increased, allowing the cell to degrade proteins and organelles and thus obtain a source of macromolecular precursors, such as amino acids, fatty acids, and nucleotides, which would not be available otherwise. Autophagy roles in cancer are a topic of intense debate. In one hand, autophagy has an anticancer role. On the other hand, when tumor cells are starved due to limited angiogenesis, autophagy stops them from dying. Autophagy is important in numerous diseases, including bacterial and viral infections, neurodegenerative disorders, several myopathies, and cardiovascular diseases. 3 Autophagy and weight loss A type of intermittent fasting is used to stimulate autophagy and to 'trick' one's metabolism into working longer hours and burning more fat. Notably, pharmacological stimulation of autophagy can reduce both weight gain and obesity-associated alterations upon hypercaloric regimens usage. Proteasomes Proteasomes are small organelles composed of protein complexes (proteases) that are responsible for proteolysis (protein breakdown) of malformed and ubiquitin-tagged proteins. Proteasomes monitor the protein content of the cell to ensure degradation of unwanted proteins, such as excess enzymes and other proteins that become unnecessary to the cell after they perform their normal functions, and malformed proteins. Protein encoded by virus should also be destroyed. The process of cytosolic proteolysis is carefully controlled by the cell, and it requires that the protein be recognized as a potential candidate for degradation. This recognition involves ubiquination, a process whereby several ubiquitin molecules (a 76-amino acid long polypeptide chain) are attached to the candidate protein using ATP. Once a protein has been marked, it is degraded by proteasomes. (Fig 2) During proteolysis, the ubiquitin molecules are released and re-enter the cytosolic pool to be re used. Fig. 3. The structure and function of the proteasome 4 Protein degradation by proteasomes in health and disease Proteins destined for degradation are labeled with ubiquitin through covalent attachment to a lysine side chain. The amino acid composition at the amino terminus determines how quickly the protein will be ubiquinated and thus the half-life of the protein. Some proteins have very long half-lives, such as the crystallins in the lens of the eye; these proteins do not turn over significantly during the human life span. Because they were synthesized largely in utero, about half the crystallins in the adult lens are older than the person. Other proteins have half-lives of 4 months (proteins such as hemoglobin that last as long as the red blood cell), or the half-life can be very short, such as for ornithine decarboxylase, which has a half-life of 11 minutes. The half-lives of proteins is influenced by the amino (N)-terminal residue, the so- called N-end rule. Destabilizing N-terminal amino acids (causing short half-life) include arginine and acetylated alanine. In contrast, serine is a stabilizing amino acid. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences) are rapidly ubiquinated and degraded and, therefore, have short half-lives Poly-ubiquination, which increases the rate of turnover/degradation of a protein, occurs by successive addition of free ubiquitin to that which is already bound to the protein. Failure of degradation of misfolded proteins by proteasomes, can lead to accumulation of abnormal proteins and development of certain diseases such as Alzheimer’s disease and Creutzfeldt–Jakob disease (Mad-cow disease). Peroxisomes Peroxisomes are small membrane bounded, self-replicating organelles. They contain more than 40 oxidative enzymes, especially urate oxidase, and D- amino acid oxidase that contain oxidative enzymes. Peroxisomes function in the catabolism of long-chained fatty acids (beta oxidation), forming acetyl coenzyme A (CoA) as well as hydrogen peroxide (H2O2) by combining hydrogen from the fatty acid with molecular oxygen. Similar to mitochondria, peroxisomes increase in size and undergo fission to form new peroxisomes; however, they possess no genetic material of their own. Inclusions Inclusions are non-living components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, pigments, and crystals. 5  1. Glycogen Glycogen is the most common storage form of glucose in human and is especially abundant in cells of muscle and liver. It appears in electron micrographs as clusters, or rosettes, of β particles (and larger α particles in the liver) that resemble ribosomes, located in the vicinity of the SER. On demand, enzymes responsible for glycogenolysis degrade glycogen into individual molecules of glucose. 2. Lipids Lipids, triglycerides in storage form, not only are stored in specialized cells (adipocytes) but also are located as individual droplets in various cell types, especially hepatocytes. Lipids are considered as potential source of energy within the cells. 3. Pigments It could be natural pigments as haemoglobin of red blood cells, melanin in the skin and hair and a yellow-to-brown pigment, lipofuscin in the long-lived cells, such as neurons and cardiac muscle. Tattoos is the injection of ink intracellular that could be phagocytosed by macrophages leading to its permanent effect. Fig. 4 Types of inclusions A. TEM of glycogen ganules in rosettes B. Lipid droplets in fat cell Clinical hint: abnormal accumulations I. Lipids 1-Steatosis (Fatty Change) Means excessive, abnormal accumulations of triglycerides within parenchymal cells due to alcohol abuse, diabetes mellitus, obesity, toxins, protein malnutrition, and anoxia 2- Cholesterol and Cholesterol Esters as in atherosclerosis. 6  I. Proteins as inAlzheimer disease. II. Glycogenas in Diabetes mellitus and Glycogen storage diseases. III. Pigments; Exogenous Carbon (coal dust),The most common air pollutant in urban areas. Its accumulation could lead to Anthracosis occurs in heavy smokers, and coal mines workers with accumulation of carbon pigment within lungs and regional lymph nodes. Endogenous Pigments Lipofuscin Patients with severe malnutrition& Cancer cachexia. Melanin: Hyperpigmentation generalized due to excessive sun exposure or localized as in benign (nevus) and malignant cutaneous tumors. Hypopigmentation Generalized as in albinism or localized as in vitiligo (autoimmune disorder). 7 22 Cytosolic Respiration : ILOs By the end of this lecture, students will be able to 1. Correlate carbohydrate intake to production of energy 2. Deduce how energy production differs among different cells and different cellular compartments 3. Correlate regulation of glycolysis to energy production ❖ What is Glycolysis? And why is it important? - Is a sequence of reactions occuring in the cytoplasm for the oxidation of Glucose to two molecules of pyruvic acid (3-carbon molecule) under aerobic conditions; or lactate under anaerobic conditions providing energy (as ATP) and intermediates for other metabolic pathways. - The unique ability to function in presence or absence of oxygen makes glycolysis the only source of energy in RBCs (as they lack mitochondria) and when performing physically- demanding tasks, the anaerobic glycolysis serves as the primary energy source for the muscles. - The glycolytic pathway may be considered as the preliminary step before complete oxidation. - It provides carbon skeletons for amino acid synthesis and the glycerol portion of fat. - It occurs as follows: Page 1 of 4  Net gain of 8 ATP produced Under anaerobic conditions NADH+H+ (at step 6) is re oxidized via lactate formation. This allows glycolysis to proceed in the absence of oxygen. Page 2 of 4  Clinical Implications: Pyruvate kinase deficiency: As mature RBCs are completely dependent on glycolysis for ATP production. Pyruvate kinase deficiency leads to decreased ATP production. This results in hemolytic anemia, with the severe form requiring regular transfusions. Severity depends both on the degree of enzyme deficiency. ❖ Regulation of the Glycolytic Pathway The regulatory enzymes of the glycolytic pathway are the 3 irreversible enzymes: hexokinase, phosphofructokinase (PFK-1), and pyruvate kinase (PK). A- Hormonal regulation: 1- Covalent Modification: - Insulin secreted in fed state activates key enzymes of glycolysis by dephosphorylation. - Glucagon secreted in fasting inhibits key enzymes of glycolysis by phosphorylation. 2- Induction/Repression: - Insulin leads to induction of key enzymes of glycolysis while glucagon represses them B- Allosteric Regulation: Page 3 of 4 Inhibitors of glycolysis: 1. Mercury inhibits glyceraldehyde-3-P dehydrogenase by binding to the enzyme’s active site. This will inhibit glycolysis to proceed leading to cell death. The most common cause of mercury poisoning is from eating polluted or wrongly preserved seafood. 2. Fluoride combines with Mg2+ as Mg fluoride, Mg is essential for the activity of enolase enzyme, therefore fluoride interferes with enolase activity. For this reason fluoride has been used for years as a rodenticide (to kill rodents) and a pesticide. It also explains why the FDA requires that all fluoride toothpastes should carry a warning that If more than used for brushing is accidentally swallowed, medical help should be seeked right away. ❖ What happens after the 2 pyruvates are produced? Pyruvate produced from glycolysis (under aerobic conditions) is then transported to the mitochondria via a special transporter where it is converted to Acetyl Co-A by “Pyruvate Dehydrogenase Complex”. (PDH) (see Aerobic VS Anaerobic). Page 4 of 4 23 The mitochondrial Structure and Citric Acid Cycle ILOs By the end of this lecture, students will be able to 1. Correlate structure of different parts of the mitochondria to their function. 2. Interpret mitochondrial response to changes in cellular activities. 3. Value the impact of mitochondrial DNA in health and disease. 4. Correlate regulation of citric acid cycle to energy production In aerobic cells, the mitochondria are the principal sites for the synthesis of adenosine triphosphate (ATP) (What is the importance of ATP?), and possess their own DNA. It imparts eosinophilia to the cytoplasm of the cell. Mitochondrial structure: Each mitochondrion possesses a smooth outer membrane and a folded inner membrane which are structurally and functionally distinct. The narrow space between the inner and outer membranes is called the intermembrane space, whereas the large space enclosed by the inner membrane is termed the matrix space (intercristal space). A. Outer Mitochondrial Membrane The outer mitochondrial membrane possesses a large number of porins, thus it is freely permeable to large molecules. As a result, the contents of the intermembrane space resemble the cytosol. Additional proteins located in the outer membrane are responsible for the formation of mitochondrial lipids. B. Inner mitochondrial membrane It encloses the matrix space and is folded into cristae to provide a larger surface area for ATP synthase and the respiratory chain. (How are the cristae affected by cellular activity?). It is relatively impermeable. The inner membrane displays the presence of a large number of lollipop- like inner membrane subunits, protein complexes known as ATP synthase, which are responsible for the generation of ATP from ADP and inorganic phosphate. The globular head of the subunit is attached to a narrow, flattened, cylinder-like stalk projecting from the inner membrane into the matrix space. Figure -1 The structure and function of mitochondria A, Mitochondrion sectioned longitudinally to demonstrate its outer and folded inner membranes. B, Enlarged inner membrane subunits and ATP synthase. C, EM mitochondria showing outer and inner membranes, cristae, matrix space and dense matrix granules (arrow heads). Page 1 of 4  C. Matrix The matrix space is filled with soluble and insoluble proteins, ribosomes, tRNA, mRNA, and dense spherical matrix granules. The matrix also contains the double-stranded mitochondrial circular deoxyribonucleic acid (cDNA) and the enzymes necessary for the expression of the mitochondrial genome. cDNA contains information for synthesis of few proteins. Therefore, most of the codes necessary for the formation and functioning of mitochondria are located in the genome of the nucleus. Clinical correlation Several diseases of mitochondrial deficiency have been described due to mutations of nuclear or mitochondrial DNA. Most of them are characterized by muscular dysfunction. Mitochondrial inheritance is maternal, because few, if any, mitochondria from the sperm nucleus remain in the cytoplasm of the zygote. Respond of mitochondria to cellular hyperactivity. The mitochondria are self-replicating (Why?) and they can generated from preexisting mitochondria. In response to hyperactivity, mitochondria enlarge in size and increase the folding of cristae and /or fuse together producing giant mitochondria (hypertrophy). Mitochondria also can replicate their DNA, and undergo fission to increase their number (hyperplasia). ❖ What is Citric Acid Cycle (CAC)? Also called tricarboxylic acid cycle (TCA) or Kreb’s cycle it is the final common pathway for complete oxidation of carbohydrates, fatty acids and many amino acids. It is an aerobic process in presence of oxygen occurring in the mitochondria. Page 2 of 4  ❖ Why is CAC important? 1- Energy production: The cycle produces 3 NADH+H+ and 1 FADH2, in the electron transport chain each NADH+H+ produces 3 ATP while each FADH produces 2 ATP, Plus 1 GTP produced at substrate level, this gives a total of 12 ATP 2- It is the final common metabolic pathway for oxidation of carbohydrates, fats and proteins, as they all supply acetyl CoA. 3- It produces intermediates important for synthesis of fatty acid and cholesterol also for synthesis of non-essential amino acids. 4- CO2 produced is used in many important reactions including different CO2-fixation reactions, purines and pyrimidines. Regulation of CAC: - The key regulatory enzymes of CAC are citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. - Primary function of the cycle is to provide energy, thus rate of the cycle is adjusted to meet the cells’ ATP demand, high levels of ATP/ADP and NADH+H+/NAD are inhibitory indicating high energy status of the cell. - ATP inhibits both citrate synthase and isocitrate dehydrogenase where as both are activated by high ADP levels. NADH+H+ inhibits isocitrate dehydrogenase and α- Ketoglutarate dehydrogenase. Page 3 of 4 - High levels of acetyl coA and oxaloacetate activate citrate synthase. - High levels of Succinyl coA inhibits α-Ketoglutarate dehydrogenase. - CAC is not covalently regulated. Page 4 of 4