Nonsense Suppression Therapy: New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes

Inherited bone marrow failure syndromes (IBMFS) are a group of cancer-prone genetic diseases characterized by hypocellular bone marrow with impairment in one or more hematopoietic lineages. The pathogenesis of IBMFS involves mutations in several genes which encode for proteins involved in DNA repair, telomere biology and ribosome biogenesis. The classical IBMFS include Shwachman–Diamond syndrome (SDS), Diamond–Blackfan anemia (DBA), Fanconi anemia (FA), dyskeratosis congenita (DC), and severe congenital neutropenia (SCN). IBMFS are associated with high risk of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. Unfortunately, no specific pharmacological therapies have been highly effective for IBMFS. Hematopoietic stem cell transplantation provides a cure for aplastic or myeloid neoplastic complications. However, it does not affect the risk of solid tumors. Since approximately 28% of FA, 24% of SCN, 21% of DBA, 20% of SDS, and 17% of DC patients harbor nonsense mutations in the respective IBMFS-related genes, we discuss the use of the nonsense suppression therapy in these diseases. We recently described the beneficial effect of ataluren, a nonsense suppressor drug, in SDS bone marrow hematopoietic cells ex vivo. A similar approach could be therefore designed for treating other IBMFS. In this review we explain in detail the new generation of nonsense suppressor molecules and their mechanistic roles. Furthermore, we will discuss strengths and limitations of these molecules which are emerging from preclinical and clinical studies. Finally we discuss the state-of-the-art of preclinical and clinical therapeutic studies carried out for IBMFS.

Yeast Sup35 Prion Structure: Two Types, Four Parts, Many Variants

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2–32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73–124, 125–153, and 154–221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2–72, which always correlated with the “strong” and “weak” [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

Yeast Sup35 prion structure: two types, four parts, many variants

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, can exist as multiple structural variants exhibiting phenotypic variation in the strength of nonsense suppression and mitotic stability. Structure of [PSI+] and its variation is only partly characterized. Here, we mapped the Sup35 proteinase K-resistant amyloid cores of 26 [PSI+] prions of different origin, isolated from yeast cells. In all cases the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153 and 154-221, but their presence differed between [PSI+] isolates. The [PSI+] phenotype depended mainly, if not solely, on the structure in region 2-72. Structures in region 73-221 were in some cases mitotically unstable and heterogenous. Two distinct digestion patterns were observed for the 2-72 fragment, which correlated with the “strong” and “weak” [PSI+] nonsense-suppressor phenotypes. All [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal in the presence of multicopy SUP35. Thus, our data suggest existence of two distinct and reliably distinguishable structural classes of [PSI+] rather than a continuum of prions with gradually altering phenotype.ImportancePrions and amyloids are relatively novel and incompletely characterized structures. To understand them better, we mapped amyloid cores of 26 isolates of the Sup35 yeast prion using proteinase K digestion and mass spectrometry. We found that these cores are composed of up to four proteinase K-resistant elements spanning almost the whole length of Sup35 region inessential for viability. However, only the N-terminal element was present in all structures. There are many variants of the Sup35 prion, and these are usually roughly combined into two groups, “strong” and “weak”, based on the strength of their nonsense-suppressor phenotype. However, it was not clear whether such groups could be distinguished by any reliable qualitative criteria. Our data indicate that these groups do exist and can be reliably distinguished based on the N-terminal element digestion pattern and the effects of the multicopy SUP35 and HSP104 genes on these prion variants.

The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI+] Prion

The [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI+] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI+], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI+] formation, while Sla2 can prevent [PSI+] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI+] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI+] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI+] detoxification system.

The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI+] Prion

The [PSI+] nonsense-suppressor determinant of Saccharomyces cerevisiae is related to formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI+] amyloids have variants in amyloid structure and in the strength of suppressor phenotype. Appearance of [PSI+], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI+] formation, while Sla2 can prevent the [PSI+] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI+] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins causes lethality of [PSI+] cells with a strong, but not with weak suppressor phenotype. This lethality results from excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow considering the Pub1 and Upf1 proteins as a novel [PSI+] detoxification system.

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

ABSTRACTNonsense suppressors (NonSups) induce “readthrough”, i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.Table of Contents artwork