Structural and Functional Consequences of a Disease Mutation in the Telomere Protein TPP1

Structural and Functional Consequences of a Disease Mutation in the Telomere Protein TPP1

Structural and functional consequences of a disease mutation in the telomere protein TPP1 Kamlesh Bishta,1, Eric M. Smitha,b,1, Valerie M. Tesmera, and Jayakrishnan Nandakumara,b,2 aDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109; and bProgram in Chemical Biology, University of Michigan, Ann Arbor, MI 48109 Edited by Joachim Lingner, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, and accepted by Editorial Board Member Dinshaw J. Patel September 29, 2016 (received for review April 8, 2016) Telomerase replicates chromosome ends to facilitate continued cell The discovery of the TEL patch of TPP1 prompted the pre- division. Mutations that compromise telomerase function result in diction that this region could be a hotspot for mutations that stem cell failure diseases, such as dyskeratosis congenita (DC). One cause telomerase-deficiency diseases, such as DC. We recently such mutation (K170Δ), residing in the telomerase-recruitment factor reported a case of a severe variant of DC, Hoyeraal–Hreidarsson TPP1, provides an excellent opportunity to structurally, biochemi- syndrome (23), in which the proband was heterozygous for a cally, and genetically dissect the mechanism of such diseases. We deletion of a single amino acid of the TPP1 protein, namely lysine ACD show through site-directed mutagenesis and X-ray crystallography 170 (K170) (24). Our study placed the gene coding for TPP1 DKC1 TERC TERT that this TPP1 disease mutation deforms the conformation of two protein on a list with 10 other genes ( , , , RTEL1 TINF2 CTC1 NOP10 NHP2 WRAP53 PARN critical amino acids of the TEL [TPP1’s glutamate (E) and leucine-rich , , , , , ,and ) that are found mutated in DC and other telomere-related dis- (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR- Δ Cas9 technology, we demonstrate that introduction of this mutation orders (25). Indeed, a heterozygous TPP1 K170 mutation was in a heterozygous manner is sufficient to shorten telomeres in human also reported in another unrelated family, where it was implicated in causing aplastic anemia and other related hematopoietic com- cells. Our findings rule out dominant-negative effects of the muta- plications in the proband (26). In both families, the presence of the tion. Instead, these findings implicate reduced TEL patch dosage in K170Δ mutation correlated strongly with short telomeres. Tran- causing telomere shortening. Our studies provide mechanistic insight sient overexpression of K170Δ in cultured human cells resulted in a into telomerase-deficiency diseases and encourage the development decrease in telomerase recruitment (24, 26), and a reduction in the of gene therapies to counter such diseases. ability of TPP1 to stimulate telomerase processivity (the ability of telomerase to continue DNA synthesis without dissociating from a telomere | telomerase | TPP1 | TEL patch | dyskeratosis congenita bound primer) (24). These results invoke a direct role for the K170Δ mutation in reducing telomerase function in dividing cells. elomerase is a unique ribonuclear protein complex [telomerase However, it is unknown whether the heterozygous K170Δ mutation Treverse transcriptase (TERT): protein subunit; TR: RNA sub- is sufficient to cause telomere shortening in dividing human cells unit] that synthesizes telomeric DNA repeats (GGTTAG in hu- without additional genetic modifiers. BIOCHEMISTRY mans) at chromosome ends to facilitate continued cell division The placement of K170 directly adjacent to E168, E169, and (1–3). Although telomerase is not expressed in most normal so- E171 strongly suggests that this amino acid facilitates the matic cells, its presence in stem cells is crucial for their self-renewal ability of the TEL patch to recognize telomerase. However, (4). Germ-line mutations in the genes coding for telomerase sub- inspection of the crystal structure of the OB domain of WT units, or in protein factors important for either telomerase bio- genesis or trafficking in the cell, result in genetically inherited Significance diseases, the most prominent of which is dyskeratosis congenita (DC) (5, 6). DC is clinically diagnosed by the presentation of a Telomerase is an enzyme that replicates chromosome ends to classic triad: nail dysplasia, skin hyperpigmentation, and oral leu- facilitate continued stem cell division. Mutations in telomerase or koplakia. Bone marrow failure resulting from the depletion of the in telomerase-related genes result in stem cell-dysfunction dis- hematopoietic stem cell pool is the primary cause of morbidity and eases, such as dyskeratosis congenita (DC). Despite its devastat- mortality in DC patients. At a molecular level, DC is diagnosed by ing nature, DC currently has no cure. Here we report the crystal blood leukocyte telomere lengths of less than the first percentile structure of a mutant protein implicated in DC to reveal how the for age. Despite the devastating nature of this disease, its genetic mutation disrupts a region of the protein essential for telomer- basis has hampered the development of effective therapies. ase function. Furthermore, we demonstrated that this mutation, The TPP1 (TINT1/PTOP/PIP1) protein (7–9) is a component when introduced into a human cell line, is sufficient to cause the of the six-membered shelterin complex [TPP1, protection of telo- cellular underpinnings of DC. Our results therefore make the meres protein (POT1), telomeric repeat-binding factor 1 (TRF1), strong prediction that correcting the mutation in the stem cells of TRF2, Rap1, and TERF1-interacting nuclear factor 2 (TIN2)] the patient will reverse the cellular symptoms of disease. that protects chromosome ends from the cellular DNA damage response and repair machineries (10, 11). TPP1 is a unique Author contributions: K.B., E.M.S., and J.N. designed research; K.B., E.M.S., V.M.T., and shelterin component in that it binds telomerase (12), recruits J.N. performed research; K.B., E.M.S., V.M.T., and J.N. analyzed data; and J.N. wrote telomerase to telomeres (13), increases telomerase processivity the paper. (14), and facilitates telomere elongation in telomerase-positive The authors declare no conflict of interest. cells (15, 16). Mutagenesis screens revealed a region called the This article is a PNAS Direct Submission. J.L. is a Guest Editor invited by the Editorial TEL [TPP1’s glutamate (E) and leucine-rich (L)] patch, which Board. resides in the structurally characterized oligosaccharide– Freely available online through the PNAS open access option. oligonucleotide-binding (OB) domain of the TPP1 protein. The Data deposition: The atomic coordinates and structure factors have been deposited in the TEL patch is critical for all of the telomerase-associated roles of Protein Data Bank, www.pdb.org (PDB ID codes 5I2X and 5I2Y). TPP1 (15–22). Central to TEL patch function are three glutamates— 1K.B. and E.M.S. contributed equally to this work. E168, E169, and E171 (Fig. 1A)—which, when mutated to ala- 2To whom correspondence should be addressed. Email: [email protected]. nine (15, 18) or arginine (17), result in severely compromised This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. telomerase function. 1073/pnas.1605685113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1605685113 PNAS | November 15, 2016 | vol. 113 | no. 46 | 13021–13026 Downloaded by guest on September 23, 2021 A C D Results Deletion of K170, but Not Its Substitution to Alanine, Greatly Reduces Telomerase Processivity. The deleterious effect of the TPP1 K170Δ mutation on telomerase processivity is not because of a defect in binding to POT1 as K170Δ binds POT1 similar to WT TPP1 protein (Fig. S1A). To further understand the molecular basis of telomerase deficiency caused by this DC mutation, we engineered B a K170A mutation in the TPP1-N vector (14) for expression in Escherichia coli. TPP1-N constructs (TPP1 amino acids 90–334) of K170A, WT, K170Δ, and E169A/E171A (EE-AA) proteins E were purified as described previously (14, 15) (Fig. S1B). We performed direct primer extension assays to determine how each of the TPP1-N variants contributes to telomerase processivity in the presence of POT1 (Fig. 1 B and C). The WT protein displayed the expected increase in telomerase processivity (14), giving rise to a large fraction of longer DNA products. Both K170Δ and E169A/ E171A failed to increase telomerase processivity to WT TPP1-N levels, as expected from previous studies (Fig. 1 B,andC) (15, 24). Strikingly, mutation of K170 to alanine resulted in only a modest reduction in telomerase processivity (Fig. 1 B and C) that was not statistically significant in experiments performed in triplicate (P = F 0.08). This result suggests that the positive charge of K170 is not critical for telomerase processivity. In addition, E169D/E171D (E→D mutation that retain negative charge), but not E169Q/ E171Q (E→Q mutations that are iso-steric and polar but lack the negative charge), was able to stimulate telomerase processivity (Fig. S1C). These results are fully consistent with the structure of TPP1–OB WT, which indicates that the glutamates, but not K170, of the TEL patch loop are accessible to telomerase. Fig. 1. The main-chain, but not the side-chain, of TPP1 K170 is important for Deletion of K170, but Not Its Substitution to Alanine, Greatly Reduces stimulating telomerase processivity, recruiting telomerase to telomeres, and Telomerase Recruitment to Telomeres and Telomere Lengthening. for telomere elongation. (A) Ribbon depiction of a part of the TPP1–OB WT Given that deletion of K170 reduces telomerase processivity but crystal structure (PDB ID code 2I46) with the TEL patch loop (amino acids substitution of K170 to alanine does not, we next asked how K170A 166–171) shown in stick representation. The protrusion in the loop is referred impacts telomerase recruitment. To this end, we engineered HeLa to here as a “knuckle.” (B) Direct primer extension assays with telomerase cell lines stably expressing either FLAG-TPP1 K170A or FLAG- extracts performed in the presence of purified POT1 (500 nM) and the in- TPP1 K170Δ protein using the Flp-recombinase–based single-site dicated TPP1-N proteins (500 nM).

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    6 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us