Specificity of ligand binding to yeast hexokinase PII studied by STD-NMR

Hexokinase catalyzes the phosphorylation of glucose and is the first enzyme in glycolysis. To investigate enzyme–ligand interactions in yeast hexokinase isoform PII under physiological conditions, we utilized the technique of Saturation Transfer Difference NMR (STD NMR) to monitor binding modes and binding affinities of different ligands at atomic resolution. These experiments clearly show that hexokinase tolerates several changes at C-2 of its main substrate glucose, whereas epimerization of C-4 significantly reduces ligand binding. Although both glucose anomers bind to yeast hexokinase, the α-form is the preferred form for the phosphorylation reaction. These findings allow mapping of tolerated and prohibited modification sites on the ligand. Furthermore, competitive titration experiments show that mannose has the highest binding affinity of all examined sugars. As several naturally occurring sugars in cells show binding affinities in a similar range, hexokinase may be considered as an ‘emergency enzyme’ in yeast cells. Taken together, our results represent a comprehensive analysis of ligand–enzyme interactions in hexokinase PII and provide a valuable basis for inhibitor design and metabolic engineering.     

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

Role of eIF3a in regulating cell cycle progression

Translational control is an essential process in regulation of gene expression, which occurs at the initiation step performed by a number of translation initiation factor complexes. eIF3a (eIF3 p170) is the largest subunit of the eIF3 complex. eIF3a has been suggested to play roles in regulating translation of a subset of mRNAs and in regulating cell cycle progression and cell proliferation. In this study, we examined the expression profile of eIF3a in cell cycle and its role in cell cycle progression. We found that eIF3a expression oscillated with cell cycle and peaked in S phase. Reducing eIF3a expression also reduced cell proliferation rate by elongating cell cycle but did not change the cell cycle distribution. However, eIF3a appears to play an important role in cellular responses to external cell cycle modulators likely by affecting synthesis of target proteins of these modulators.     

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.     

日本血吸虫真核翻译起始因子5基因的cDNA克隆和免疫保护功能分析

利用抑制削减杂交法筛选日本血吸虫雌雄虫差异基因,从中获得真核翻译起始因子5基因(eIF5)的 表达序列标。对该序列进行生物信息学分析:对其5’端进行电子延伸,获得含完整开放阅读框的cDNA序列 (AY686501)。将其亚克隆到表达载体pET-28c,进行重组表达。免疫保护效果实验表明,重组表达蛋白具有显著 抑制血吸虫卵在寄主肝脏中的沉积效果。     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

白细胞介素2亲和性配体的筛选

白细胞介素2(IL-2)及其受体拮抗剂的研究对免疫抑制药物的研制具有重要作用.抗白细胞介素2受体α链中和性单克隆抗体5G1(抗Tac型抗体)能够特异性地阻断IL-2与其受体的结合.因此,5G1可作为目标分子被用来在噬菌体展示肽库中筛选白细胞介素2的亲和性配体.经过4轮亲和性筛选以及5G1亲和活性的测定,6个具有明显5G1亲和活性的噬菌体克隆被发现.DNA序列分析结果显示出,所得到的肽序列具有明显的保守性,即SSFT(L/P)I.该序列与IL-2受体α链没有同源性.因此,SSFT(L/P)I可能模拟了IL-2受体α链上的一个不连续表位(mimotope),为白细胞介素2亲和性配体片段.     

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A)

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.