Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

• Keywords
• Trypanosoma cruzi, differentiation, eIF2, phosphorylation, translation, virulence,
• MeSH
• Cell Line MeSH
• Chagas Disease parasitology   MeSH
• CRISPR-Cas Systems MeSH
• Eukaryotic Initiation Factor-2 genetics   metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Parasitemia MeSH
• Proteome metabolism   MeSH
• Protein Biosynthesis MeSH
• Protozoan Proteins analysis   biosynthesis   metabolism   MeSH
• Gene Expression Regulation MeSH
• Life Cycle Stages MeSH
• Trypanosoma cruzi growth & development   metabolism   pathogenicity   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-2 MeSH
• Proteome MeSH
• Protozoan Proteins MeSH

The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

• Keywords
• ISR, S. cerevisiae, biochemistry, chromosomes, eIF2, eIF2B, genes, protein interactions, translational control,
• MeSH
• Eukaryotic Initiation Factor-2 genetics   MeSH
• Eukaryotic Initiation Factor-2B genetics   MeSH
• Phosphorylation MeSH
• Proteins MeSH
• Protein Biosynthesis MeSH
• Publication type
• Journal Article MeSH
• Comment MeSH
• Names of Substances
• Eukaryotic Initiation Factor-2 MeSH
• Eukaryotic Initiation Factor-2B MeSH
• Proteins MeSH

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• Keywords
• ATF4, GCN4, Ribo-seq, TCP-seq, UTR, co-translational assembly, eIF2, eIF3, gene expression, mRNA, ribosome, ribosome profiling, translational control,
• MeSH
• 5' Untranslated Regions MeSH
• Eukaryotic Initiation Factor-2 genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Peptide Initiation Factors genetics   metabolism   MeSH
• Codon, Initiator MeSH
• Humans MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Activating Transcription Factor 4 genetics   metabolism   MeSH
• Basic-Leucine Zipper Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5' Untranslated Regions MeSH
• ATF4 protein, human MeSH Browser
• Eukaryotic Initiation Factor-2 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• GCN4 protein, S cerevisiae MeSH Browser
• Peptide Initiation Factors MeSH
• Codon, Initiator MeSH
• Multiprotein Complexes MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Activating Transcription Factor 4 MeSH
• Basic-Leucine Zipper Transcription Factors MeSH

Vascular calcification (VC) is an independent risk factor for cardiovascular events and all-cause mortality with the absence of current treatment. This study aimed to investigate whether eIF2alpha phosphorylation inhibition could ameliorate VC. VC in rats was induced by administration of vitamin D3 (3×10(5) IU/kg, intramuscularly) plus nicotine (25 mg/kg, intragastrically). ISRIB (0.25 mg/kg·week), an inhibitor of eIF2alpha phosphorylation, ameliorated the elevation of calcium deposition and ALP activity in calcified rat aortas, accompanied by amelioration of increased SBP, PP, and PWV. The decreased protein levels of calponin and SM22alpha, and the increased levels of RUNX2 and BMP2 in calcified aorta were all rescued by ISRIB, while the increased levels of the GRP78, GRP94, and C/EBP homologous proteins in rats with VC were also attenuated. Moreover, ISRIB could prevent the elevation of eIF2alpha phosphorylation and ATF4, and partially inhibit PERK phosphorylation in the calcified aorta. These results suggested that an eIF2alpha phosphorylation inhibitor could ameliorate VC pathogenesis by blocking eIF2alpha/ATF4 signaling, which may provide a new target for VC prevention and treatment.

• MeSH
• Aorta metabolism   MeSH
• Cholecalciferol metabolism   MeSH
• Eukaryotic Initiation Factor-2 * MeSH
• Phosphorylation MeSH
• Rats MeSH
• Vascular Calcification * chemically induced   drug therapy   prevention & control   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cholecalciferol MeSH
• Eukaryotic Initiation Factor-2 * MeSH

Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.

• Keywords
• ES6(S), ES7(S), ES9(S), Trypanosoma cruzi, cryo-EM, eIF1, eIF2, eIF3, eIF5-CTD, k-DDX60, the 43S pre-initiation complex, translation initiation,
• MeSH
• Models, Molecular MeSH
• Protein Biosynthesis immunology   MeSH
• Mammals MeSH
• Trypanosomatina pathogenicity   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

MEHMO syndrome is a rare X-linked syndrome characterized by Mental retardation, Epilepsy, Hypogenitalism, Microcephaly, and Obesity associated with the defect of protein synthesis caused by the EIF2S3 gene mutations. We hypothesized that the defect in protein synthesis could have an impact on the immune system. We describe immunologic phenotype and possible treatment outcomes in patient with MEHMO syndrome carrying a frame-shift mutation (I465fs) in the EIF2S3 gene. The proband (currently 9-year-old boy) had normal IgG and IgM levels, but had frequent respiratory and urinary tract infections. On subcutaneous immunoglobulin therapy achieving supra-physiological IgG levels the frequency of infections significantly decreased in Poisson regression by 54.5 % (CI 33.2-89.7, p=0.017). The MEHMO patient had had frequent acute infections despite normal IgG and IgM serum levels and responded well to the immunoglobulin treatment.

• MeSH
• Child MeSH
• Epilepsy drug therapy   genetics   immunology   pathology   MeSH
• Eukaryotic Initiation Factor-2 genetics   MeSH
• Phenotype MeSH
• Hypogonadism drug therapy   genetics   immunology   pathology   MeSH
• Humans MeSH
• X-Linked Intellectual Disability drug therapy   genetics   immunology   pathology   MeSH
• Microcephaly drug therapy   genetics   immunology   pathology   MeSH
• Mutation * MeSH
• Obesity drug therapy   genetics   immunology   pathology   MeSH
• Genitalia abnormalities   immunology   pathology   MeSH
• Treatment Outcome MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Names of Substances
• EIF2S3 protein, human MeSH Browser
• Eukaryotic Initiation Factor-2 MeSH

Recently, the genetic cause of several syndromic forms of glycemia dysregulation has been described. One of them, MEHMO syndrome, is a rare X-linked syndrome recently linked to the EIF2S3 gene mutations. MEHMO is characterized by Mental retardation, Epilepsy, Hypogonadism/hypogenitalism, Microcephaly, and Obesity. Moreover, patients with MEHMO had also diabetes and endocrine phenotype, but detailed information is missing. We aimed to provide more details on the endocrine phenotype in two previously reported male probands with MEHMO carrying a frame-shift mutation (I465fs) in the EIF2S3 gene. Both probands had a neonatal hypoglycemia, early onset insulin-dependent diabetes, and hypopituitarism due to dysregulation and gradual decline of peptide hormone secretion. Based on the clinical course in our two probands and also in previously published patients, neonatal hypoglycemia followed by early-onset diabetes and hypopituitarism may be a consistent part of the MEHMO phenotype.

• MeSH
• Diabetes Mellitus, Type 1 congenital   genetics   MeSH
• Endocrine Glands metabolism   MeSH
• Epilepsy genetics   MeSH
• Eukaryotic Initiation Factor-2 genetics   MeSH
• Phenotype MeSH
• Hypoglycemia congenital   genetics   MeSH
• Hypogonadism genetics   MeSH
• Hypopituitarism congenital   genetics   MeSH
• Humans MeSH
• X-Linked Intellectual Disability genetics   MeSH
• Microcephaly genetics   MeSH
• Infant, Newborn MeSH
• Obesity genetics   MeSH
• Genitalia abnormalities   MeSH
• Frameshift Mutation MeSH
• Transcription Factors MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• EIF2S3 protein, human MeSH Browser
• Eif2s3y protein, mouse MeSH Browser
• Eukaryotic Initiation Factor-2 MeSH
• Transcription Factors 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.

• Keywords
• S. cerevisiae, biochemistry, biophysics, eIF3, initiation, mRNA recruitment, ribosome, structural biology, translation, yeast,
• 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
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Guanosine Triphosphate MeSH
• RNA, Messenger MeSH
• Mutant Proteins MeSH
• Protein Subunits MeSH
• RNA, Transfer, Met MeSH

AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

• Keywords
• ER stress, Lewy body disease, Parkinson's disease, alpha‐synuclein, unfolded protein response,
• MeSH
• alpha-Synuclein * metabolism   MeSH
• Biomarkers metabolism   MeSH
• Endoplasmic Reticulum Chaperone BiP * metabolism   MeSH
• Lewy Body Disease * pathology   metabolism   MeSH
• Eukaryotic Initiation Factor-2 * metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Brain metabolism   pathology   MeSH
• Nerve Growth Factors metabolism   MeSH
• Heat-Shock Proteins * metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Unfolded Protein Response * physiology   MeSH
• Endoplasmic Reticulum Stress physiology   MeSH
• Up-Regulation * MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alpha-Synuclein * MeSH
• Biomarkers MeSH
• Endoplasmic Reticulum Chaperone BiP * MeSH
• EIF2S1 protein, human MeSH Browser
• Eukaryotic Initiation Factor-2 * MeSH
• HSPA5 protein, human MeSH Browser
• MANF protein, human MeSH Browser
• Nerve Growth Factors MeSH
• Heat-Shock Proteins * MeSH
• SNCA protein, human MeSH Browser

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
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Codon, Initiator MeSH
• Multiprotein Complexes MeSH
• NIP1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH