Nejnovější poznatky v biologii chronické lymfocytární leukemie (CLL) mají významný dopad na léčbu tohoto onemocnění. Buňky CLL vykazují závislost na zvýšené expresi BCL-2, autonomní BCR signalizaci a vyznačují se nadměrnou expresí delta izoformy p110 PI3K kinázy a BTK kinázy, která podporuje přežívání nádorových buněk. Cílená léčba monoklonálními protilátkami rituximabem a obinutuzumabem spolu s malými molekulami, jako je ibrutinib, idelalisib a venetoklax, výrazně rozšířila terapeutické možnosti, což vedlo ke zlepšení celkového přežití pacientů. V této souvislosti je pozoruhodné, že pacienti, kteří zahájili léčbu ibrutinibem, vykazují míru přežití srovnatelnou s celkovou populací. Ne všechny problémy spojené s CLL však byly vyřešeny, neboť stále přetrvávají otázky týkající se dysfunkce imunitního systému a sekundárních malignit, jednotnosti léčebných přístupů pro pacienty s vysokým a nízkým rizikem, dlouhodobých strategií pro mladé pacienty a terapie pro pacienty s Richterovou transformací. Přestože inhibitory BTK a BCL-2 mohou pozitivně ovlivnit imunitní systém, problémy spojené s infekcemi a sekundárními nádory přetrvávají. Pokud jde o Richterovu transformaci, identifikace specifických genetických abnormalit může v budoucnu umožnit cílenější a účinnější léčbu, včetně terapie CAR-T a bispecifických protilátek.
New findings in the biology of chronic lymphocytic leukemia (CLL) have major implications for the treatment of this disease. CLL cells exhibit a dependence on increased expression of BCL-2, an autonomous BCR signaling pathway, and are characterized by overexpression of the p110 PI3K delta kinase isoform and BTK kinase, which promotes tumor cell survival. Targeted therapies such as the monoclonal antibodies rituximab and obinutuzumab, along with small molecules such as ibrutinib, idelalisib, and venetoclax, have dramatically expanded therapeutic options, resulting in improved overall patient survival. In this context, it is noteworthy that patients starting treatment with ibrutinib have survival rates comparable to the general population. However, not all issues have been resolved, as there are questions regarding the immune system, consistency in treatment approaches and long-term strategies for young patients, especially those with Richter transformation. Although BTK and BCL-2 inhibitors can positively influence the immune system, we still face challenges related to infections and secondary tumors. Regarding Richter transformation, identification of specific genetic abnormalities may allow more targeted and effective therapies in the future, including CAR-T therapy and bispecific antibodies.
- Keywords
- Richterova transformace,
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell drug therapy genetics pathology MeSH
- Immune System immunology pathology MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Agammaglobulinaemia Tyrosine Kinase antagonists & inhibitors MeSH
- Antineoplastic Agents administration & dosage therapeutic use MeSH
- Antineoplastic Protocols MeSH
- Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors MeSH
- Check Tag
- Humans MeSH
The 12-subunit mammalian eIF3 is the largest and most complex translation initiation factor and has been implicated in numerous steps of translation initiation, termination and ribosomal recycling. Imbalanced eIF3 expression levels are observed in various types of cancer and developmental disorders, but the consequences of altered eIF3 subunit expression on its overall structure and composition, and on translation in general, remain unclear. We present the first complete in vivo study monitoring the effects of RNAi knockdown of each subunit of human eIF3 on its function, subunit balance and integrity. We show that the eIF3b and octameric eIF3a subunits serve as the nucleation core around which other subunits assemble in an ordered way into two interconnected modules: the yeast-like core and the octamer, respectively. In the absence of eIF3b neither module forms in vivo, whereas eIF3d knock-down results in severe proliferation defects with no impact on eIF3 integrity. Disrupting the octamer produces an array of subcomplexes with potential roles in translational regulation. This study, outlining the mechanism of eIF3 assembly and illustrating how imbalanced expression of eIF3 subunits impacts the factor's overall expression profile, thus provides a comprehensive guide to the human eIF3 complex and to the relationship between eIF3 misregulation and cancer.
Standard screening of melanoma patients is a useful tool for predicting outcome of patients, however, an instant methodology for exact detection of subclinical disease or monitoring treatment response is under investigation. Detection of circulating melanoma cells is, therefore, a possible novel promising staging method. However, inconsistent data on method sensitivity and on the predicted patient outcome has been shown repeatedly. Recently, a multimarker real-time RT-PCR methodology for quantification of five melanoma markers Melan-A, gp 100, MAGE-3, MIA and tyrosinase was described by our group. In the current prospective trial, blood specimens of 65 patients with AJCC stage IIB-III cutaneous melanoma after surgery were periodically examined. In the above group, 27 % of subjects relapsed during the study. Prior to the disease progression we could observe a statistically significant tumor marker elevation in previous 0 to 9 months in all patients with clinical relapse. MAGE-3 became the most sensitive progression marker. During progression, three concordant positive markers were seen in 39 % of patients, followed by two concordant positive markers in 28 % and 1 marker in 33 %. This study supports the use of a multimarker real-time RT-PCR as a disease progression predictor. The dynamic assessment of serially obtained blood specimens represents a useful method for early metastasis detection and treatment response of melanoma patients.
- MeSH
- Early Diagnosis MeSH
- Adult MeSH
- Eukaryotic Initiation Factor-3 genetics MeSH
- Financing, Organized MeSH
- Genetic Markers MeSH
- Genetic Testing methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Melanoma diagnosis genetics MeSH
- Cell Line, Tumor MeSH
- Skin Neoplasms diagnosis genetics MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Controlled Clinical Trial MeSH
In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.
- MeSH
- Eukaryotic Initiation Factor-3 genetics metabolism MeSH
- Peptide Chain Initiation, Translational physiology MeSH
- Codon, Initiator genetics metabolism MeSH
- Ribosome Subunits, Small, Eukaryotic genetics metabolism MeSH
- Multiprotein Complexes genetics metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.
- MeSH
- Eukaryotic Initiation Factor-3 chemistry genetics metabolism MeSH
- Protein Conformation * MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Models, Molecular MeSH
- Protein Subunits chemistry genetics metabolism MeSH
- Protein Biosynthesis * MeSH
- Ribosomes genetics metabolism MeSH
- Saccharomyces cerevisiae Proteins chemistry genetics metabolism MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cells have elaborated a complex strategy to maintain protein homeostasis under physiological as well as stress conditions with the aim to ensure the smooth functioning of vital processes and producing healthy offspring. Impairment of one of the most important processes in living cells, translation, might have serious consequences including various brain disorders in humans. Here, we describe a variant of the translation initiation factor eIF3a, Rpg1-3, mutated in its PCI domain that displays an attenuated translation efficiency and formation of reversible assemblies at physiological growth conditions. Rpg1-3-GFP assemblies are not sequestered within mother cells only as usual for misfolded-protein aggregates and are freely transmitted from the mother cell into the bud although they are of non-amyloid nature. Their bud-directed transmission and the active movement within the cell area depend on the intact actin cytoskeleton and the related molecular motor Myo2. Mutations in the Rpg1-3 protein render not only eIF3a but, more importantly, also the eIF3 core complex prone to aggregation that is potentiated by the limited availability of Hsp70 and Hsp40 chaperones. Our results open the way to understand mechanisms yeast cells employ to cope with malfunction and aggregation of essential proteins and their complexes.
- MeSH
- Eukaryotic Initiation Factor-3 genetics MeSH
- Humans MeSH
- Actin Cytoskeleton genetics MeSH
- Mitochondria MeSH
- Mutation MeSH
- Myosin Type V genetics MeSH
- Protein Aggregates genetics MeSH
- HSP40 Heat-Shock Proteins genetics MeSH
- HSP70 Heat-Shock Proteins genetics MeSH
- Saccharomyces cerevisiae Proteins genetics MeSH
- Saccharomyces cerevisiae genetics growth & development MeSH
- Myosin Heavy Chains genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.
- MeSH
- Eukaryotic Initiation Factor-1 genetics MeSH
- Eukaryotic Initiation Factor-3 chemistry genetics metabolism MeSH
- Phenotype MeSH
- Gene Dosage MeSH
- Peptide Chain Initiation, Translational MeSH
- Codon, Initiator MeSH
- Crystallography, X-Ray MeSH
- Yeasts genetics growth & development MeSH
- Ribosome Subunits, Small, Eukaryotic metabolism MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Saccharomyces cerevisiae Proteins chemistry genetics metabolism MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.
Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.
- MeSH
- Eukaryotic Initiation Factor-3 chemistry metabolism MeSH
- Peptide Chain Initiation, Translational * MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Open Reading Frames * MeSH
- Protein Biosynthesis MeSH
- Ribosomes metabolism MeSH
- Mammals MeSH
- Activating Transcription Factor 4 chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.
- MeSH
- Eukaryotic Initiation Factor-3 genetics metabolism MeSH
- Guanosine Triphosphate metabolism MeSH
- RNA, Messenger metabolism MeSH
- DNA Mutational Analysis MeSH
- Mutant Proteins genetics metabolism MeSH
- Protein Subunits genetics metabolism MeSH
- Protein Biosynthesis MeSH
- Ribosomes metabolism MeSH
- RNA, Transfer, Met metabolism MeSH
- Saccharomyces cerevisiae genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
