PROTEOMICS Cellular proteins and peptides

1

Genes and Proteins. Introduction to genetic Engineering

1.1 Genes and genome

Gene¹ is the unit of inheritance and the unit of genetic information, made by polynucleotide sequences. The totality of individual genes constitutes the genome. Genes in humans, intended as tracts of DNA that can be transcribed in the final mRNA and further translated in proteins, constitute as little as 5 percent of the genomic DNA. Human genome has been sequenced in the late ‘90s. The most impactant surprise was that only about 23 000 genes – about the same number of Drosophila melanogaster and the plant Arabidopsis, barely more than the 1000 cells-formed worm Caenorhabditis elegans, half than the rice, Oryza saliva, about 10 000 less than water flea – are contained in the 5.5 billion base pairs that make up human DNA. So few coding genes (less than 2 percent of the entire human genome) codify for the program to build up about 90 000 estimated proteins, including the proteins that regulate gene functions, metabolism, signaling, cell division, defense. Most of the remaining non-coding nuclear DNA in eukaryotes is occupied by diverse families of repeats (introns see 1.11, mobile elements see 1.4, and large intergenic regions), including arrays of apparently non-specific satellite sequences and transposons (see 1.4) that constitute the epigenetic elements of the genome (see 1.8). Introns (see 1.11), non-codifying sequences inside genes, account for about one third of the human genome. A large part of formally called non-coding DNA is devoted to codify for regulatory RNA. Promoters (see 1.8) are DNA segments located at the head of genes. Pseudogenes (see 4.1) are inactive genes originated from the original ones by mutations, duplication, processing or retrotransposition (see 1.4). Their number in genome is quoted about the same as the codifying genes. Pseudogenes generate small RNAs (sRNA, see 1.8 and 8.0) that have regulatory functions. MicroRNA (mRNAs), see 1.8, are small, non coding genes that are found in the genomes of most eukaryotes, where they play an important role in gene expression regulation.

Regulatory proteins, RNA (including sRNAs, see 1.8), non-coding bits of DNA, even chemical and structural alterations of the genome itself control how, where, and when genes are expressed. Alternate splicing (see 1.11) and other post-transcriptional events multiply the potential number of proteins that a single gene may transcribe.

The body’s complete set of proteins corresponding to the genome sequence is defined proteome, and proteomics is the branch of biology that studies the proteins in a cell or tissue.

In eukaryotic cells, genome is contained in a nucleus surrounded by cytoplasm. Prokaryotic cells have no nuclei to contain the genome.

Based on genome structure, living beings are distinct in three categories:

•  VIRUSES: the viral genome is a single-or double-stranded DNA or RNA, with few tenths of genes. Replication depends on the mechanisms of the host cell. Phagi are viruses replicating in bacteria, with linear double-stranded DNA.

•  PROKARYOTES: possess only one circular chromosome formed by a double-helix DNA, normally in aploid state, without dominant or recessive genes. Bacterial DNA occupies the central part of the bacterial cell, referred to as the nucleoid, and is associated to nucleoid-associated proteins (NAPs) that perform two major functions: gene regulation and chromosome organization. Reproduction is generally asexuate, operated by cell division (mitosis). Genes are few thousands. Nucleus and mitochondria are absent. Some circular, extrachromosomal DNA can be found in cytoplasm. These ring structures are called plasmids. Bacterial genomes have intronless coding regions aligned almost contiguously along the chromosome. Pseudogenes (nonfunctional genes), lateral gene transfer regions, and transposable elements may be present. According to genome size, a distinction in free living bacteria (5-10 Mbases), recent or facultative pathogens (2-5 Mb), and obligate symbionts or pathogens (2-5 Mb) can be made.

•  EUKARYOTES: have a genome distributed in many chromosomes. In Caenorhabditis elegans, a six-chromosome nematode whose genome has been fully sequenced², 97 × 10⁶ bases are present, codifying for over 19 000 genes.

Somatic cells are generally diploids, germinal cells are in aploid state. Each gene may exist in several alternate forms, or alleles. By mating, alleles from maternal and paternal origin are recombined in diploid arrangement. The genetic constitution of an organism marks the genotype which is responsible for creating the phenotype. Reproduction is generally sexuated with gene recombination (meiosis). Meiosis is a type of cell division that reduces the double set of chromosomes (diploid state of alleles in eukaryotes) to a single set (aploid state), as observed in germinal cells (see 1.6 under Meiosis and germ...).

Gene recombination is distinct in several forms:

•  Crossing-over or general recombination allows favorable and unfavorable mutations to be separed, providing a mean to escape and to spread over for favorable alleles;

•  Conservative or site-specific recombination is responsible for the integration of a phagic genome into bacterial chromosomes;

•  Replicative recombination is related to the ability of certain genome elements to move from one location to another (transposable elements in bacteria).

Gene recombination is normal in eukaryotes and exceptional in prokaryotes, in which three recombination mechanisms are possible:

•  Sexuate recombination transfers all or part of the genetic material from a donor (+) to an acceptor (−) cell through connecting bridges;

•  Bacterial transformation, in which exogenous DNA is incorporated into the chromosome of a receptor strain. In eukaryotic cells, the incorporation of exogenous nucleic acids is called transfection;

•  Transduction transports genes (transductible genes) to an acceptor cell by means of a phage or a plasmid.

In viruses, gene recombination takes place by hybridation, when an host cell is simultaneously infected by two viruses (viral recombination).

In experimental conditions, hybridation is obtained by manipulation of two different eukaryotic cells. Hybrid cells obtained by these techniques are hybridomas.

1.2 Genome structure

In eukaryotic cells, three different forms of DNA are found: in chromosomes (I), in mitochondria (II), and in extra-nuclear space (III).

I - Genomic or chromosomal DNA is formed by paired sequences of nucleotides (nucleobases + deoxyribose + phosphate) held in chain by 3-5’ phosphodiester bonds (fig. 1.1). Nucleobases are two purines (adenine A and guanine G) and two pyrimidines (cytosine C and thymine T). Nucleobase sequences determine the aminoacid sequences (primary structure) of the proteins according to the triplet rule. Each triplet is a codon. Since 64 triplets can be obtained by combining 4 nucleobases, while the aminoacids in natural proteins are only 20, there is quite a redundancy in genetic code, with possibly several triplets codifying for one aminoacid (degeneracy) and some nonsense triplets acting as starting and ending signals for transcription (tab. 1.1, where U = uracil in RNA substitutes T in DNA).

In form of double helix, two DNA strands are tied together by hydrogen bonds according to the rule of complementary base pairing that states: in a double-strand (duplex) structure the hydrogen bonds are always established between A and T (two bonds) and between C and G (three bonds). Therefore for the duplex DNA in chromosomes the following three stoichiometric relations, already noted by Chargaff and utilized to build up the Watson and Crick’s model, are valid:

1. A = T, G = C;

2. A + G (total purines) = T + C (total pyrimidines);

3. A + T/G + C: is characteristic for each species.

Tab.1.1 Triplet (codon) meaning*

Double-stranded DNA is in open (linear) or closed (ring) structure. Rings are usually supercoiled in a superhelix and in this shape are unable to replicate, except after the rupture of one of the supercoiled chains. In vitro, both prokaryotic and eukaryotic RNA polymerases (see 1.8 and 1.9) can initiate the transcription more efficiently when the template is supercoiled.

Bacterial DNA gyrases introduce negative supercoils, while topoisomerase I (see 1.9) relaxes negative supercoils. Endonucleases break one chain in specific points with consequent superhelix unfolding. Replication takes place only on one of the complementary strands after separation of the duplex along an initiation tract by action of releasing proteins known as swivelases. Experimentally, the same result can be obtained by insertion of ethidium bromide.

Chromosomal DNA is twisted around basic proteins called histones. The total DNA length of a single chromosome in Drosophila melanogaster is 4 cm, MW = 80 × 10⁹ Da (80 MDa). In each nucleus of mammalian cells, total chromosomal DNA length is about 2 meters, containing about 4-5.5 × 10⁹ nucleobases!

II - Mitochondrial DNA (mtDNA) is a double-stranded, circular, supercoiled DNA with MW of about 10 × 10⁶ Da, like the viral DNA in bacteriophages. In humans, the base pairs for each ring are heredited exclusively from the mother’s egg cell. Each ring of mtDNA contains the information for 37 proteins: 13 genes codify for subunits of ATP-synthetase and for complexes I to IV of the respiratory chain (see 3.8) and 24 genes are involved in the production of transfer and ribosomal RNA (tRNA and rRNA, see 1.10) and in control functions. Sometimes, mtDNA is organized in peculiar structures called chain dimers. Superhelix release is operated by endonucleases, swivelases or ethidium bromide, as mentioned.

Curiously, animal mitochondrial genomes, unlike nuclear genomes of eukaryotes, are essentially free of noncoding DNA (see 1.1), whereas plant mitochondrial genome is full of junk DNA.

III - Extra-nuclear DNA is involved in cytoplasmic inheritance not regulated by mendelian rules (epigenetics, see 1.8).

During the interphase of cell cycle, chromosomes are indistinct, embedded in an heterogeneous material termed chromatin. Chromatin is a tangle of DNA, histones, non-histone basic proteins (protamines, see 8.6), acidic proteins, histone-modifying and chromatin-remodeling enzymes that act during replication, nuclear RNA, and lipids. Non-histone chromosomal protein family includes the 30-kDa members of High mobility group (HMG) that represent late mediators of endotoxin-induced release of macrophagic factors (TNF, IL-1)³. Histone- and chromatin-involved enzymes are, for instance, the ATRX ATPase belonging to SNF-2 family, and the multifunctional protein DAXX that associate to ATRX in an active complex operating in chromatin remodeling, in H3-3 histone deposition in nucleosomes and in telomere stabilization, see 1.7.

In chromatin, double-stranded DNA and histones are folded in ring-shaped, supercoiled (therefore inactive) structures called nucleosomes. Each nucleosome includes 2 coils of DNA (about 200 nucleobase pairs) around an octamer of histones. Nucleosomes are distributed as collier grains or clustered along the chromosomal DNA. Euchromatin is the chromatin region where DNA transcription (see 1.9) is activated. The compact chromatin regions where genes are silent are called heterochromatin. In these regions, the compact structure unpairs the access to the elements that permit gene transcription. Chromatin structure is dynamically changed by mechanisms of epigenetic modulation (see 1.8) that include chromatin remodeling and histone modifications such as acetylation, methylation, phosphorylation by specific enzymes (see herebelow).

Drosophila genome (four chromosomes) is about 180 Mb (megabases) in size, a third of which is centric heterochromatin consisting primarily of simple repeated sequence satellites, transposons and two large blocks of ribosomal RNA genes. The euchromatin portion of the genome, about 120 Mb encoding about 13 600 genes, has been sequenced⁴.

Histones are 10-20 kDa basic proteins divided in 5 classes, H1, H2a, H2b, H3, H4. Beside their support functions for twisted DNA in nucleosomes, they contribute in directing the sequential binding of factors that: 1-unfold the superhelix and the chromatin tangle; 2-partially detach repressor histones; 3-modulate the activation of the regulator and operator regions; 4-favor the binding of RNA polymerase⁵.

Hystone-modifying enzymes (transferases as HAT, see 1.8; kinases) mark hystones in specific sites by methylation (chromodomains), acetylation (bromodomains) or phosphorylation. Other enzymes (demethylases, deacetylases like sirtuins, see 1.7, phosphatases) switch off the added groups. The same imprinting is observed in DNA and RNA regions. Post-translational histone modifications such as phosphorylation, acetylation, and methylation regulate a broad range of DNA- and chromatin-templated nuclear events, including transcription (see 1.9).

Bacterial DNA is normally contained in one single big circular chromosome coiled in a superhelix with MW of about 2 × 10⁹ Da and in additional 1 to 20 rings of double-stranded DNA, called plasmids. The dimensions of each plasmid are similar to mitochondrial and viral DNA. The number of plasmids remains constant during replication. Sometimes, extra-chromosomal DNA structures (episomes) form side loops that may be integrated in the chromosome.

Episomes and plasmids in bacteria are carriers of tracts of genic information such as sex markers, bacteriokin expression (see 8.6 under other factors of innate immunity) and genes for the resistence to antibiotics. During sexuate replication, plasmids are transferred from a donor to an acceptor cell.

Viral genome is DNA or RNA in single or double chain (see 16.1). Molecular weight of viral genome varies from 2 to 200 × 10⁶ Da. Viral DNA can be transiently present in linear twin chains but derives from, or is converted into ring structures. Indeed, special sequences are frequently found at the double chain endings, permitting the ligation in ring structures or the integration of viral genome into the genome of the host cell (lysogeny, see 16.3), according to complementary base pairing. These sequences (see Figure 1.3) are described as:

Sticky ends: one ending is located at, or added to the 3-end of one chain, and the complementary sequence is located at the 5’-extremity of the second chain. Ring ligation is performed by DNA ligases;

Terminal repetitions added to both the 3-ends by terminal transferases. Then ligation takes place with the complementary terminal sequences of another tract of duplex DNA, provided that the complementary sequence exists or is introduced by nucleases;

Circular permutations: families of linear DNA originating the same sequences when annealed in rings by combined action of esonucleases and DNA ligases. Sticky ends and terminal repetitions, generated by specific endonucleases or introduced by manipulation, are used in genetic engineering in order to obtain hybrid cells by recombinant rDNA technics (see 1.12 and fig. 1.3).

1.3 Some chemical and physical properties of DNA

In order to isolate nuclear material in form of double or single strands, to observe DNA in electron microscopy or to obtain sufficient amounts of a peculiar DNA for further studies, some DNA properties are utilized in a preliminary separation and characterization, namely:

 I.    A linear relation exists between DNA density in CsCl gradient centrifugation and percentage content of C-G pairs, since they are connected by three hydrogen bonds instead of two, as in A-T pairing. Therefore the C-G pairing confers a more compact and dense structure;

 II.   Heat produces DNA denaturation by sharp stretching of the double helix and separation of the twin strands. Consequently, a rapid increase in absorbance at 260 nm occurs, as a consequence of the exposition of purine and pyrimidine rings previously embedded in the double helix structure. Urea and alkali also induce strand separation. Stretching point is the temperature at which the increase in absorbance is observed;

III.   A linear relation exists between stretching point and percentage of C-G pairing, since A-T pairs, with only two hydrogen bonds, are less stable and easier to be separed by heat. A similar denaturation is obtained by submitting DNA solutions to alkaline conditions. Denatured regions are made evident by electron microscopy;

IV.   Pairing of complementary strands or tracts, either with DNA or with RNA, is easily obtained by simple mixing of the neutral solutions.

When a nucleotide sequence is known, for instance by means of sequential analysis (see 2.3), it can be synthetized. By this procedure, labeled nucleobases can be incorporated. The labeled sequence will pair to the complementary region of a single-stranded DNA, forming a double helix tract (probing) that may be revealed by autoradiography or by electron microscopy. The duplex may be isolated from single-stranded DNA by methods based on differences in molecular mass (blotting). Methods based on the use of polynucleotide chains having a known sequence (probes) are widely used in sequential studies leading to definition and isolation of genes.

The genetic approach to locate genes in chromosomes is termed chromosomal mapping. Normal or mutant genetic material is recombined and transfered from cell to cell. Then the modifications induced in the phenotype of the acceptor cells and their progeny are registered.

The search for specific defects and mutations in genes is performed by means of amplification (Polymerase Chain Reaction, see 1.12 and 2.3) followed by blotting (Southern blotting) of DNA sequences. By analogy, normal or defective RNA sequences can be analyzed by Northern blotting. Defective proteins expressed by genes are identified by means of specific antibodies (Western blotting).

1.4 Spontaneous and induced mutations. Mutagens.

Mutations occur spontaneously or may be induced by chemical or physical agents (mutagens). Natural or induced mutant genes can be introduced in embryonal experimental animals, producing transgenic (carriers of an altered gene) or knock-out (lacking a gene) strains of animals that represent one of the most important tools in biogenetical studies. The knowledge of the specific altered gene and observations of the effects induced by the alteration permit to correlate the gene to the functions of the product generated by. The structural modifications in genoma sequences induced by mutations affect one single nucleobase (point mutations) or entire tracts of nucleic acids. Point mutations occur by transition, transversion, insertion or deletion of nucleobases:

Transition: one purine is substituted by another purine (for instance, G for A), or one pyrimidine by another pyrimidine, so as the base pairing is changed. Transitional mutations occur spontaneously or are induced by agents such as nitrous acid that transforms adenine in hypoxantine (adenine pairs to thymine while hypoxantine pairs to cytosine);

Transversion: one purine is substituted by one pyrimidine and viceversa, or a base mispairing occurs as a consequence of introduction of nucleobase analogs as 5-bromouracil and 5-aminopurine. Transversion is common in spontaneous mutations, e.g., more than 50 percent of mutations affecting the α and β chains in hemoglobin (see 3.8) are transversions;

Insertion of a couple of nucleobases. Acridine insertion between two subsequent bases causes pairing to a complementary base in the second strand leading to shifting of the triplet sequence;

Deletion of a couple with consequent shifting. Lytic agents cause mutations by deletion.

Mutations by transition and transversion are relatively harmless since they are easily eliminated by endogenous mechanisms of repair. On the contrary, mutations that insert or delete a single base will change the reading frame for the entire subsequent sequence (frame shift). The shift must be corrected by repair processes (see 1.7) or driven to cell apoptosis (see 1.7), except for insertion or deletion of three (or multiple of three) nucleotides in sequence.

Genetic information can be altered by enzymatic modification of nucleotide sequences, a process named editing. In mammals, editing enzymes are two classes of nuclear and mitochondrial deaminases that deaminate encoded adenine (A) to form inosine (I) or encoded cytidine (C) to uridine (U): U couples to A, I couples to C in the template DNA strand, producing nonfunctional tracts of double stranded DNA. Besides the roles played in neurotransmission and lipid metabolism, editing enzymes may fulfill essential roles in the defense against infectious agents as mediators of antibody diversification (see 8.2) and as potent intracellular poisons for viral replication (see 16.1)⁶.

Many chemical and physical agents induce non-point mutations, including D-lysergic acid diethylamide (LSD), methylated purines, UV, X, γ radiations. Expansion of certain nucleotide trios, such as CAG repeats, each coding for glutamine Q, or CGG coding for glycine G, in coding or noncoding sequences involves more than 30 human diseases. The CAG repeat causes polyQ diseases such as progressive hereditary chorea or HUNTIGTON’S DISEASE, and SPINOCEREBELLAR ATAXIA. CGG expansion originates the FRAGILE X SYNDROME, the most common cause of mental retardation next to Down syndrome. Most of UV-induced mutations affect two adjacent molecules of thymine that become tied in thymine dimers fairly easily removed by repair mechanisms. Insertion of stretches of additional material, including transposons or transposable elements (repetitive DNA sequences that can jump about the genome) are quite frequent.

Transposons can expand the genome, resulting in repetitive DNA sequences, but they can also contribute to substantial DNA losses. High density of transposable elements and DNA repeats characterize the constitutive heterochromatin of eukaryotic chromosomes (the name is given because heterochromatin stains differently from euchromatin where the genes are collected). The juxtaposition of heterochromatic regions, transmissible in epigenetic forms, with euchromatic genes results in gene silencing regulation and, perhaps, contributes to chromosome segregation at the centromere and to cell division.

Retrotransposons, considered to make up about 40 % of the mammalian genome, are duplicated by a copy and paste mechanism (a small piece of DNA is transcribed in RNA, reverse-transcribed into DNA and the complementary DNA is inserted back at a new site in the genome) instead of the cut and paste (splicing) mechanism without intermediate RNA used by most of the transposable elements.

Cut and paste and copy and paste are peculiar cases of the general processes of splicing and alternate splicing (see 1.11). A class of retrotransposons called Alu elements, short tracts of genomic DNA ending in a poly-A tail, is contained in the human genome in 1.4 millions copies. It has been calculated that one new Alu copy is produced and inserted all over into the genome each 100-200 births.

Retrotransposons are classified as those that bear long terminal repeats (LTRs) also known as endogenous retroviruses, and those that do not (L1 elements, or LINE-1, SINE, Alu). They participate in genome modeling, however they also represent genetic threats through insertional mutagenesis, translocations and other rearrangements by recombination, aberrant transcription from their promoter, antisense effects. Retrotransposon control is operated via cytosine methylation of CpG dinucleotides (major mechanism) or via cytidine deamination by editing enzymes (see 1.11).

Insertion of heterologous DNA in bacteria or cells may be considered an induced non-point mutation. Bacteria react to the insertion by recognition and attack. In fact, bacteria possess a set of enzymes that label its own DNA, such as methylases, able to methylate adenine groups that are adjacent to specific sequences of genome.

Restriction endonucleases (see 2.3) operating inside a bacterium are able to split the exogenous DNA in the same positions where the cellular DNA has been modified by methylation. Splitting is operated after recognition of specific sequences on genome. DNA is split on both strands and therefore cannot be repaired anymore. DNA tracts that are modified by methylation are made not recognizable by bacterial endonucleases. This is a form of innate immunity that is present in prokaryotes (see 8.0). Some phagi possess methylases cheating the restriction endonucleases of the host cell and allowing viral DNA to escape their attack.

Restriction enzymes permit to operate on genomic material and to build up restriction maps that identify a linear series of sites cleaved by digestion with different endonucleases, separed by the actual distance along the nucleic acid. Genetic maps identify a series of sites at which mutations occur. The distance between the sites is determined by recombination frequences. The restriction maps of the wild type and of the corresponding mutant DNA permit to relate the restriction maps to genetic maps.

Locations along the chromosomes where a single base varies among different people are called single-nucleotide polymorphisms (SNP or snips).

Mutagens and mutations may act on germ line cells. In this rare event, mutations are amplified to somatic, generally lethal mutations. Except for particular cases, like thalassemia that is favored in regions where malaria is spread, also potentially advantageous mutations will be rejected by the environmental drift. Also artificial hybrids would be eliminated if not protected by acting on the surrounding environment.

Point mutations affecting somatic cells, an event lasting for the life span, act on genes and genome with alternate issues:

•  the single cell whose genome undergoes a point mutation transcribes an altered protein, but this production may be insignificant in front of the huge amount of normal cells producing the original protein;

•  an overload of point mutations in different specialized cells may be involved in the general phenomenon of senescence;

•  point mutations affecting a silent gene are uneffective until the gene is silent;

•  a point mutation affecting a triplet which corresponds to an aminoacid that is irrelevant for the overall activity of the transcribed protein causes only a progressive accumulation of the isoform from the mutated clone. The appearence of isoforms sometimes counteracts the environmental drift and represents an advantageous way to substitute or to differentiate gene-depending substances (valid also for germ line cells);

•  cells contain the repair information to delete a mutation (valid also for germ line cells);

•  genetic code redundance in some instances permits a mutated triplet to transcribe the same aminoacid (valid also for germ line cells);

•  but when a mutation affects specific points of peculiar genes called protooncogenes, the last becomes an oncogene giving origin to transformed cells.

1.5 Cellular and viral oncogenes

⁷

The common feature of oncogenes⁸ is to be altered forms of normal genes, proto-oncogenes, that partecipate to a proliferation program entering into the network of genes codifying for factors that receive the growth signal and transduce it to activate effector genes. Oncogene deregulation and overactivity lead the cell to proliferative disfunctions and to a block of apoptosis (see 1.7), a typical picture of cancer. A central event in oncogenesis is the formation of specific protein aggregates that normally serve as signaling units for cellular events but can also operate as inappropriate signals for growth and metastatic abilities of cancer cells.

Proto-oncogenes normally express proteins that are transcriptional modifiers (activators or repressors) of normal cellular processes, such as Growth Factors (GF) and their receptors (GF-R), see 9.1, Guanine nucleotide-binding Proteins (G proteins, see 15.2), Protein Kinases (PK, see 15.4), Nuclear Transcription Factors (TF, see 1.8) and Adaptor Proteins (AP, see 7.4).

Oncogenes can originate also by insertion of viral nucleic material into the genome of host cells: Retroviruses (RNA viruses) and many DNA viruses are carriers of oncogenes and potential transforming agents. Therefore they are also called tumor viruses. The mechanism by which retroviral genome is incorporated in host cells is described in 16.1.

Oncogenes derived by mutation of proto-oncogenes or transmitted by viruses express oncoproteins that are able to transform normal cells to malignancy. In other cases the proteins expressed by proto-oncogenes and oncogenes are the same, but the transforming activity of the corresponding oncoprotein is due to its presence where it normally is absent or to its expression at a much higher level than normal. Some oncogenes are herebelow described:

•  fms oncogene. The acronyme derives from McDonough feline sarcoma virus (MFSV). It is a mutant of the proto-oncogene codifying for Macrophage Colony-Stimulating Factor (M-CSF or CSF-1), see 12.4 ;

•  sis oncogene, found in simian sarcoma virus (SSV), encodes a type of Platelet-Derived Growth Factor (PDGF), see 9.1, that may transform cells naturally bearing the PDGF receptor ;

•  neu oncogene found in neuroblastoma cells, derives from a proto-oncogene by mutation of a single nucleobase (guanine G for thymine T) in a critical DNA sequence of 350 bases included in the main sequence of more than 5000 bases. The neu proto-oncogene produces a transmembrane Epidermal Growth Factor Receptor (EGFR) with a Tyr-PK (Y-PK) activity in the cytosolic domain. The mutant receptor expressed by neu oncogene, in which Val is substituted for Gln in the transmembrane region, continues to exert PK activity even in absence of ligand. The human analog of the murine neu is the oncogene Her-2 that means Human EGF Receptor;

•  erbB oncogene codifies for another EGFR in which the extracellular ligandbinding domain is deleted, while erbA codifies for a modified receptor of thyroid hormone (THR). Both erbA and erbB, first recognized in avian erythroblastosis virus (AEV), act synergistically to express full oncogenicity in the target cells;

•  c-mpl oncogene (c stays for cell) and its proto-oncogene codifying for a receptor of MK-CSF and other haematopoietic cytokines, will be described in 12.4;

•  c-cbl oncogene codifies for protein ligases of the E3 complex (see 1.6) that shut down the signaling of activated GFR tyrosine kinases (see 9.0) by inducing their ubiquitination. Many artificial genes have been created for Growth Factors (for instance, for TGF) and for Colony Stimulating Factors such as GM-CSF. When these genes are introduced in cells bearing the appropriate receptor, the expressed protein continuously stimulates cell growth. Such self-stimulation in absence of a signal is called autocrine induction of cell proliferation;

•  src and abl (the acronymes are derived from Rous avian sarcoma and Abelson murine leukemia viruses, where they were formerly observed) encode a non-receptor cytoplasmic Y-PK. The mutant Y-PK lacks the N-terminal myristoylated Gly (see 3.3) that is present in normal forms. Therefore the mutant Y-PK can no longer adhere to the plasma membrane and acts as an unspecific phosphorylating enzyme in cytoplasm also in absence of signals. Like Src and Abl, many other viral oncoproteins are Y-PKs that phosphorylate at high levels different substrate proteins and induce the transformed state. Many oncoproteins, including c-Src and c-Abl, possess the SH2 and SH3 domains of adaptor proteins, see 7.4 (SH stands for Src homologies);

•  crk oncogene, found in avian sarcoma virus (ASV), codifies for an adaptor protein binding Paxillin (see 7.1) to guanine nucleotide exchange factors associated to G proteins (see 15.2);

•  ras oncogenes (see 1.7) were the first non-viral oncogenes to be discovered, found in neuroblastoma and in leukemias (c-N-ras), in lung and colon carcinoma (c-Ki-ras), see 3.3 and in bladder, mammary and skin carcinomas (cHa-ras). v-Ha-ras was found in Harvey murine sarcoma virus (HMSV) and v-Ki-ras in Kirsten murine sarcoma virus (KMSV). The prefix c stays for cell, whereas v stays for virus. The proto-oncogenes corresponding to ras codify for the GTPase subunit of Guanine nucleotide-binding proteins (G proteins, see 15.2), identified with the nuclear protein p21 (see 1.6). Prenylated Ras oncoproteins (see 3.3) are continuously maintained in an active state and initiate the cell transformation from normal to malignant. Only one mutation, Valfor Gly in position 12 of the sequence, is sufficient to transform the proto-oncogene product in a Ras oncoprotein. Ras mutants have been found in 30-50 % of people suffering of lung and colon carcinoma and in over 90 % of pancreas carcinomas. Moreover, RAS-activating mutations result in developmental defects, with or without predisposition to tumors (Costello and Noonan syndromes; cardio-facio-cutal syndrome). Also germline mutations affecting negative RAS regulators such as neurofibromin (NF1) and p120GAP (see 7.4) can originate type I neurofibromatosis and capillary malformation, respectively;

•  c-jun oncogenes codify for c-jun nuclear Transcription Factors (TF, see 1.8). Jun and Fos proteins form the active dimer of the adaptor protein and transcription factor AP see 7.4, involved in growth and development. c-Myc functions at a critical decision point of cell growth to favor proliferation and to block terminal differentiation.⁹ Myc is commonly overexpressed in human and rodent osteogenic sarcomas. The mutant Jun, Fos and Myc oncoproteins presumably activate the transcription of key genes that encode growth-promoting proteins or are more stable forms operating at unregulated levels.

Tumoral cells often contain chromosome translocations, fused elements from different chromosomes, as well as deletions, amplifications, and inappropriate numbers of chromosomes (aneuploidy). In some human tumoral cells, c-abl or c-myc have been found at points of chromosomal translocations, as seen in many cases of BURKITT’S LYMPHOMA. Identification of the genetic alterations displayed by human solid tumors as secondary events in tumor development, including oncogenic fusions and tandem gene duplications, is the goal of cancer genomic research (see App.B). This is illustrated by the discovery of an oncogenic fusion between bcr and abl genes in CHRONIC MYELOUS LEUKEMIA, as well as a genetic fusion of braf oncogene with an uncharacterized gene called KIAA154g in children with PILOCYTIC ASTROCITOMA. Another typical translocation between chrosomes 15 and 17 is the fusion of the gene incording retinoic acid receptor α (RARα) and the gene pml producing promyelocyte cells instead of mature neutrophils. In fact, the fusion protein PML-RARα blocks myeloid differentiation. Identification of genetic alterations displayed by human solid tumors as secondary events in tumor development, including oncogenic fusions and tandem gene duplications, is the goal of cancer genomic research (see App. B).

The cooperative action of oncogenes operating in cytoplasm as ras, and at nuclear level as myc, leads to transformed immortalized cell lines.

Oncosuppressors, such as genes coding for Retinoblastoma nucleoprotein (Rb), Anaphase-promoting Complex (APC, see 1.6), and p53, present on the nuclear surface (see 1.7), prevent transcription errors and point mutations to accumulate by slowing down the cell cycle. This permits the repair process to operate or drives the cell towards a programmed death (see 1.7). Mutations in oncosuppressors can be oncogenic and have been found in most types of human cancer. A modified Rb form has been found in retinoblastoma (hence, the name). Some oncoproteins, such as MDM2, normally acting as p53 down-regulators, are p53 inhibitors when overexpressed. P53 is found to be inactive in about half of all cancer cases¹⁰ (see 1.7).

MDM2 is an E3-ubiquitin ligase with a RING finger motif. MDM stays for Murine Double Minute Chromosome, referred to its discovery by genomic amplification.

Cells are maintained in a quiescent state by oncosuppressors of the Rb family that maintain factors of the E complex (notably E2F, see 1.7) in inactive (complexed) form. As soon as cyclins (see 1.6) split the Rb/E2 complex following signaling from outside, E2F enters the nucleus and starts mitosis, as described in 1.6. Mutations in Rb hamper the formation of the Rb/E2 complex, a concomitant cause of uncontrolled mitosis in retinoblastoma and other tumors. Alternatively, mutation-induced overexpression of c-myc genes stimulates the expression of Id2 protein that competes with Rb, hampering the formation of the Rb/E2 in-active complex. This mechanism is operative in the early onset of NEUROBLASTOMA in children¹¹.

The identification of oncogenes (more than 100) and oncosuppressors (at least 15) led to formulate an axiome in cancer research: the multistage process of tumor formation is driven by progressive acquisition of activating mutations in recessive growth-enhancing genes (oncogenes or cancer genes) and inactivating mutations in recessive growth-inhibitory genes (oncosuppressors) or by epigenetic abnormalities leading to increased or decreased expression of these genes¹².

1.6 Cell cycle and cycle block

¹³

The entry of cells into the cell cycle from a quiescent state and the progression of cells around the cycle are precisely controlled events. Cyclins, cyclin-dependent kinases, p53 and Rb check-point regulators are all elements of this control system. At the heart of this mechanism is anaphase - promoting complex / cyclosome (APC/C, see herebelow), whose activity is modulated by an activator as Cdc 20, see below, or by an inhibitory complex called Mitotic Checkpoint Complex (MCC). This control system permits eukaryotic cells to line up and to segregate their chromosomes during cell division providing a fail-safe mechanism. Altered expression of at least one regulatory element triggers an oncogenic event.

In humans, cells are about 10¹⁴ of about 250 different types and during the average life span they undergo 10¹⁶ divisions or cell cycles. The normal cycle of a cell whose genome is affected by an error, a mutation or a damage must be delayed at DNA integrity checkpoints before or during the repair processes, usually in early metaphase. Then repair is performed (transient, p53-independent arrest) or the cell is driven to apoptosis or programmed death (prolonged or permanent, p53-dependent, arrest, see 1.7). The steps in cell cycle are described in sequence was G0/G1 -S-G2 -M phase, where G0/G1 is referred to resting cells and pre-DNA synthetic phase (days), S is the period of DNA and sister chromatide synthesis (chromosome replication, 2-4 hrs), G2 is the premitotic interval (chromosome segregation, tetraploid state, 2-4 hrs) where DNA repair takes place, and M (1-2 hrs) is the period of mitosis that includes prophase, metaphase, anaphase, telophase. Checkpoints are placed at G1 -S-G2 phases. In yeast undergoing the mitotic process cohesin, a multisubunit complex, involved in many nuclear events, is essential for sister chromatid cohesion for much of interphase during DNA replication and maintains this association until the onset of anaphase. Cohesin, called chromosome glue, binds the sister chromatides during metaphase, opposing the spindle action across kinetochores until the anaphase-promoting complex (APC), a metaphase checkpoint bound to and activated by protein Cdc20, permits chromatide segregation and poleward migration, activating the cell anaphase. Other regulatory proteins (Mad2, Mps1), acting as downstream spindle checkpoints during anaphase, supervise the mitotic process.

Another complex, condensins, having ATPase activity, is necessary for mitotic chromosome condensation at the end of interphase. Condensins are members of the SMC (structural maintenance of chromosomes) family and consist of an SMC dimer and non-SMC subunits, including a consensus sequence and an ATP-binding cassette (ABC, see 10.6). They bind DNA in such a way to permit it to be partitioned in repetitive, stable structures¹⁴. Chromosomal DNA condensation (e.g., folding of DNA into nucleosomes in eukaryotes¹⁵, see 1.2) is essential, both in eukaryotes and in prokaryotes, for cell division because compact DNA is much easier to be split between two daughter cells than DNA in its expanded form. Much more must be understood on DNA condensation in eukaryotes, in which condensins, topoisomerases (see 1.9) and other DNA-binding proteins must be involved.

Many homologs of proteins making mitotic complexes in budding yeast are found also in fungi, Drosophila, vertebrates, including humans.

The early cell cycle transitions are regulated by transcription factors belonging to the E2F (E2F-1 to E2F-5 and E2F-6) and Myc families. E2F, in turn, are regulated by oncosuppressor nuclear Retinoblastoma nucleoproteins (Rb, p107, p130), see 1.5, in such a way that one Rb, when bound to E2F, maintains E2F target genes in a transcriptionally inactive state corresponding to the G stages (longterm quiescent G0 and transiently quiescent stage G1). As soon as E2F-bound Rb are phosphorylated by cyclin-dependent kinases (see below), active E2F is released from the Rb/E2Fcomplex and the cell cycle proceeds on. A peculiar role is played by E2F-6 that assembles the chromatin-modifying complex made by histone methyltransferases and acetylases (see 1.2), members of the Myc family (Mga/Max), proteins of the Polycomb group (PcG), and the transcriptional repressor HP1γ for long-term transcriptional silencing.

Normal mitosis from prophase to telophase is regulated by Cyclin-dependent Kinases (CDKs) in activated form.

Cyclins¹⁶ were originally defined as proteins that are specifically degraded at every mitosis. This is still true for several types of cyclins, having an approximate half-life of 20 min. According to the present knowledge, cyclins are proteins containing homologous sequences in a 100-aminoacid region called cyclin box involved in binding a Protein Kinase (CDK) partner. Two other regions are peculiar of cyclin structure: the N-terminal region called destruction box where the cyclins bound in Cyclin/CDK complexes are marked for degradation, and a PEST region (see 1.8) responsible for the short life.

Destruction of mitotic cyclins at or shortly before sister chromatid separation is operated by the ubiquitin-protein ligase Anaphase-promoting Complex (APC), also called cyclosome.

APC destroys also a class of anaphase-inhibitory proteins, securins, that maintains in an inactive state a group of caspase-like endopeptidases (thiol proteases of the CD subclass, see 1.7 and tab. 4.3) called separins. Separin activation triggers cohesin cleavage, sister chromatid separation and transition from metaphase to anaphase.

In mammalian cells, transcription of many cyclins is absolutely dependent on the presence of Growth Factors (GFs, see 9.1). Therefore cyclin synthesis, as a growth factor sensor, links cell cycle progression to external cues.

Oocyte maturation is regulated by two activities described as Maturation Promoting Factor (MPF) and Cytostatic Factor (CSF) that are able respectively to induce immature oocytes to mature so as to be ready for fertilization (see 6.6) and to arrest the unfertilized eggs at metaphase.

MPF is a complex of the universal M-phase kinase Cdc2 in humans, the product of one of the genes involved in silencing apparatus (see