Zinc Fingers of Death

Andrey Panov, "Biomolecule"

A protein complex called shelterin binds to telomeric repeats and protects them from degradation. However, it has recently been discovered that it has a competitor with a less mild character: the TZAP protein with eleven zinc fingers is able to specifically bind to the telomeres of chromosomes, but not to protect, but to trim them. Under normal conditions, he helps the cell, but if you give free rein to his fingers, TZAP will thoroughly "pinch" the telomeres, and this can lead the cell to suicide.

Diagram of the structure of typical protein motifs "zinc fingers". Most often, a zinc finger consists of 20 amino acids. The zinc ion is bound to two histidines and two cysteines. Drawings from the website studopedia.su and from Wikipedia.

For the normal functioning of eukaryotic cells, the length of telomeres – the end sections of chromosomes - is very important. Telomeres consist of short DNA repeats (TTAGGG) and almost 200 proteins that perform many important functions – from DNA synthesis to telomere protection [1].

Until January 2017, only one protein complex specifically interacting with telomere DNA repeats was known - shelterin. But now scientists have discovered another protein that has an affinity for TTAGGG sites. Due to special DNA-binding motives (see the title picture), this protein, depending on the conditions, can kill the cell, and can save it [1].

Telomeres and telomerase

In eukaryotes, special structures called telomeres are formed at the ends of chromosomes. They protect the DNA of chromosomes from degradation by nucleases and various aberrations. The DNA of telomeres consists of short conservative tandem repeats (Fig. 1). In vertebrates, for example, the six–nucleotide TTAGGG motif is repeated thousands of times, in insects – TTAGG, in most plants - TTTAGGG, in fungi the length of the motif varies greatly [2].

Figure 1. Chromosome and telomere. The sequence of telomeric repeats of the infusoria is shown. Drawing from the website gazeta.ru , adapted.

Unfortunately, when the genetic material is doubled, the cell is unable to ensure complete replication of telomeres. Therefore, after each division, the daughter cell has chromosomes with slightly shorter telomeres. This is called terminal underreplication, which, along with other mechanisms of telomere shortening, is considered as one of the causes of aging [3]. It is not yet clear how significant a role telomere reduction plays in human aging, but it is obvious that it leads to the replicative aging of cells and their transition to a senescent state. At the same time, the cell loses its ability to divide and secretes a special set of substances that form a pro-inflammatory microenvironment unfavorable for neighbors and the body as a whole.

In mammals, the average length of telomeres in a young cell is 8-12 thousand pairs of nucleotides (etc. n). And the critical (minimum allowable) length of the human telomere is 77 nucleotides. Further cell divisions can lead to extremely unfavorable events for it, including the fusion of the ends of different chromosomes [4].

In most eukaryotes, the required telomere length is maintained by telomerase, which makes up for the repeats lost during replication at the 3’ end of DNA. The complementary chain is completed by DNA polymerase [3].

In 2009, Elizabeth Blackburn, Carol Greider and Jack Shostak were awarded the Nobel Prize in Physiology or Medicine for the discovery of a mechanism that protects chromosomes from terminal under-replication using telomeres and telomerase.: "The "ageless" Nobel Prize: in 2009, works on telomeres and telomerase were awarded" [5]. Interesting information on the telomeric problem itself can be found in the articles "Aging – payment for the suppression of cancer tumors?" and "Telomeres and new targets of proto-oncogenic therapy" [6, 7]. Studies are also interesting, for example, on changes in telomere length due to the time of year in plants or during the rainy season in Costa Ricans: "Telomere length and seasons" [8]. – Ed.

Telomerase is a ribonucleoprotein complex consisting of a telomerase reverse transcriptase (TERT) enzyme and a telomerase RNA component (TERC), which contains a matrix sequence for telomere elongation – 3’-AAUCCC-5’. In one "sitting", telomerase can add not one, but several telomeric repeats at once (Fig. 2). During the sequential transition of the enzyme from state 1 through states 2 and 3 to state 4, telomerase adds one telomeric repeat to the primer. Transition 4→2 corresponds to the addition of several telomeric repeats without separation from the telomere (transition 4→1) [3].

Figure 2. The work of telomerase. The numbers within the framework indicate the position of telomerase in relation to the primer (3’-end of the DNA of the telomere) at various stages: 1 – the enzyme is not bound to the primer; 2 – annealing of the primer; 3 – stage of elongation; 4 – completion of the addition of one telomeric repeat. The dotted arrows show the possible processes of dissociation of the primer during the operation of the enzyme. Symbols: TERT is a protein subunit with a telomeric DNA anchoring site (gray circle), TERC is a telomerase RNA with a matrix site (gray rectangle). Figure from [3].

Telomerase is not active in most human somatic cells. Although the RNA component is transcribed at a constant level in almost all cells, the protein part, reverse transcriptase, is not synthesized in somatic cells. With the artificial activation of the expression of its genes, the culture of somatic cells avoids replicative aging, that is, cells acquire the ability to divide indefinitely. Telomerase collects and works in stem, germ, and some other types of cells that need to be constantly divided (for example, intestinal epithelial cells) [9, 10].

It is believed that the activation of this enzyme is associated with the development of cancer: telomerase is active in 85% of cancerous tumors, in the remaining 15% there are alternative mechanisms for maintaining telomere length based on recombination [11, 12].

Shelterin

Shelterin, or telosome, is a complex of six proteins that regulates telomerase activity and protects mammalian telomeres from DNA repair systems (Fig. 3). By binding to TTAGGG repeats on telomeric DNA, shelterin promotes the formation of a t–loop at its end - a kind of "cap" hiding the free end of the chromosome from repair enzymes. The absence or critical deficiency of shelterin in the cell "prints" telomeres, and they become available to nucleases and other enzymes, undergo destruction and merge with the ends of other chromosomes, which eventually leads to cellular senescence or apoptosis [13, 14].

Figure 3. Shelterin. a – Subunits of shelterin, their structure and sites of interaction with DNA. b is a diagram of a complex assembled on telomeric DNA. Figure from [15].

At the very end of the telomeres of humans and other warm-blooded animals, there is a rather long, 30-300 nucleotides, section of single-stranded DNA with a free 3’-end. It is to this thread that the shelterin subunit POT1 attaches, which hides the free 3’-end from telomerase and nuclease (Fig. 4). In this case, POT1 can attach not only to repeats at the end of the telomere, but also to internal repeats [15].

Figure 4. Operation of the telosome. a is the structure of the t-loop. The overhanging 3'-end is wound between two DNA chains, forming a D-loop ("substitution loop", where one of the two main DNA chains is displaced by an invading chain, complementary to the second main chain). The loop size is variable. b is a model of loop formation by shelterin. TRF1 and TRF2 bend the telomere and fix the t-loop. POT1 holds single-stranded sections of DNA. b is a model of telomere length regulation by shelterin. As long as the telomere is long enough, shelterin closes telomerase access to it. As soon as the telomere shortens to a certain length and the attached shelterins become insufficient to form a t-loop, telomerase gains access to the open 3’-end. Figure from [15], adapted. If the ends of telomeric DNA remain free, repair systems are activated that recognize such ends as double-stranded breaks.

For example, a deficiency in the cell of the shelterin subunit TRF2 can lead to the "baring" of the ends. But at the same time, in such an open state, telomeres are an excellent substrate for the work of telomerase, which completes them to the necessary, stabilizing length [14].

A new protein discovered by Eros Lazzerini Denchi and Julia Su Jo Lee (Fig. 5) – scientists from the Scripps Research Institute in the USA – along with shelterin plays an important role in the homeostasis of chromosomal telomeres. Scientists have named it the zinc finger telomeric protein (TZAP, telomeric zinc-finger associated protein).

Figure 5. Professor Denchi and student Li. Photo from the website scripps.edu .

A distinctive feature of TZAP is the 11 zinc fingers with which it "grabs" the TTAGGG repeats of telomeres. At the same time, it turned out that for effective interaction with DNA, the protein needs only the last three fingers, Znf9–11 (Fig. 6). Binding to DNA, TZAP initiates a "haircut" of telomeres: six-nucleotide repeats are cut out of them [1].

Figure 6. The last three fingers are required by TZAP to attach to telomeric DNA. On the left are Variants of the TZAP protein: whole, with eight first fingers (Znf1–8) and with the last three (Znf9–11). On the right is an experiment on the interaction of three TZAP variants with the telomere. Only the whole protein and the protein with the last three fingers bind to DNA. Figure from [1].

TZAP attaches to telomeres both in cells with working telomerase and in cells without it. At the same time, the protein does not need to interact with the components of shelterin [1].

TZAP prefers to join long telomeres. This was shown in an experiment with HeLa cells: one of their lines contained telomeres with a length of 5 t.p.n., the second – 20 t.p.n. TZAP connected with telomeres of the second line and showed no interest at all in telomeres from the first. It turned out that the amount of shelterin (including its TRF2 subunit) in the cell is constant and does not depend on the length of telomeres. Therefore, TTAGGG repeats free of TRF2 can be "exposed" on long telomeres. It is to them that TZAP reaches out with his fingers. Moreover, it competes for the substrate with TRF2, and not with TRF1. With increased expression of the TRF2 gene, the amount of TZAP attached to DNA decreases [1].

At the same time, in cells with increased expression of the TZAP gene, deprived of telomerase (as in ordinary somatic cells), chromosomes rapidly lost telomeres (Fig. 7). As a result, cells with chromosomes appeared without telomeres at all. Apparently, at a high concentration, TZAP pushes TRF2 into competition, getting the opportunity to grab hold of repetitions with your fingers and "trim" the telomere "to zero". And this leads to sad consequences: chromosomal aberrations, cell senescence and apoptosis [1].

Figure 7. Experiment with TZAP overexpression in cells without telomerase. a – Chromosomes in a normal cell (left) and in a cell with increased TZAP expression (right). Green dots mark telomeres. b is the percentage ratio of the number of chromosomes with telomere–free ends in both variants. Figure from [1].

In cells with a normal TRF2/TZAP balance, the latter is only allowed to make sure that the telomere does not become too long. TZAP also performs this function in embryonic stem cells: during experimental deletion of TZAP genes, telomeres in stem cells were significantly lengthened, and after the introduction of exogenous TZAP, they returned to normal [1].

It is known that too long telomeres can contribute to the transformation of a cell into a cancerous one, allowing it to divide more than it should. It turns out that TZAP, by regulating the maximum length of telomeres, participates in protecting the body from the occurrence of tumors [1]. But if the synthesis of TRF2 is suddenly disrupted, TZAP's hooked fingers will immediately reach for telomeres to cut, cut and cut... Until cellular death.

"These cell clocks must be very precisely tuned to allow cells to divide enough times, developing differentiated tissues of the body and supporting renewable ones, and at the same time preventing the appearance of malignant cells" (Professor Denchi) [16].

Literature

1. Li J.S., Miralles Fuste J., Simavorian T., Bartocci C., Tsai J., Karlseder J., Lazzerini Denchi E. (2017). TZAP: a telomere-associated protein involved in telomere length control. Science. doi: 10.1126/science.aah6752;
2. Wikipedia: "Telomeres";
3. Zvereva M.E., Shcherbakova D.M., Dontsova O.A. (2010). Telomerase: structure, functions and ways of regulating activity. Advances in biological chemistry. 50, 155–202;
4. Capper R., Britt-Compton B., Tankimanova M., Rowson J., Letsolo B., Man S. et al. (2007). The nature of telomere fusion and a definition of the critical telomere length in human cells. Genes Dev. 21, 2495–2508;
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13. Wikipedia: "Shelterin";
14. Panero J., Santos P.D., Slavutsky I. (2017). Telomere protein complexes and their role in lymphoid malignancies. Front. Biosci. (Schol. Ed.). 9, 17–30;
15. de Lange T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110;
16. TSRI scientists discover master regulator of cellular aging. (2017). The Scripps Research Institute.

Portal "Eternal youth" http://vechnayamolodost.ru  31.01.2017