Structure-Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin.

Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart's molecular characteristics.

A new crystal form of human histidine triad nucleotide-binding protein 1 (hHINT1) in complex with adenosine 5'-monophosphate at 1.38†Šresolution.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5'-monophosphate (AMP) complex at 1.38 Šresolution obtained from a new monoclinic crystal form is reported. The final structure has R(cryst) = 0.1207 (R(free) = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.

High-resolution X-ray structure of the rabbit histidine triad nucleotide-binding protein 1 (rHINT1)-adenosine complex at 1.10†Šresolution.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine-triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the first complete structure of the rabbit HINT1-adenosine complex is reported at 1.10 Šresolution, which is one of the highest resolutions obtained for a HINT1 structure. The final structure has an R(cryst) of 14.25% (R(free) = 16.77%) and the model exhibits good stereochemical qualities. A detailed analysis of the atomic resolution data allowed an update of the details of the protein structure in comparison to previously published data.

Histidine triad nucleotide-binding protein 1 (HINT-1) phosphoramidase transforms nucleoside 5'-O-phosphorothioates to nucleoside 5'-O-phosphates.

Nucleoside 5'-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5'-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5'-O-monophosphorothioate (AMPS) to 5'-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2'-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS ≥ CMPS > UMPS > dAMPS ≫ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released.

[HINT1--a novel tumor suppressor protein of the HIT superfamily]. / HINT1--nowy supresor nowotworów w rodzinie bialek HIT.

The histidine triad nucleotide binding protein1 (Hint1) belongs to the first branch of the HIT superfamily. Hint1 catalyses the process of hydrolysis of the P-N bond in AMP-lysine, AMP-alanine, AMP-NH2. The physiological role of this enzyme is still unclear. There is accumulating evidence that HINT1 is a novel tumor suppressor protein, albeit the mechanism of action of HINT1 in respect to tumor suppression is not fully understood. Recent findings have shown that Hint1 inhibits the activity of the transcription factors AP1, MITF and USF2, as well as influences the transcription process of some genes of Wnt/beta-catenin pathway. Thereby, it seems that Hint1 exerts its major cellular function as gene transcription regulator, and thus, this function provides its potential role as a tumor suppressor protein. The clinical relevance of impairments in the Hint1 expression with the respect to specific human cancers is still a matter of extensive studies.