NMR Characterization of Angiogenin Variants and tRNAAla Products Impacting Aberrant Protein Oligomerization

Abstract

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation.

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases

“…In conclusion, it could be worth evaluating in the future if ANG self-association, although not being massive, might affect residues or domains crucial for the intracellular routing of the protein, and/or affect for instance ANG action in the stress granules [ 32 ]. This scenario might, in fact, help the onset and/or development of the aforementioned neurodegenerative diseases.…”

“…Despite expressing many positive functions, ANG can favour the growth of solid tumors by inducing neovascularization around the malignant tissue mass [ 16 ], while some ANG mutants are implicated in neurodegenerative diseases, because their dysfunction often leads to motor neurons hypoxia and death [ 31 ]. Many variants arising from pathogenic mutations influencing the ANG structure; substrate binding and therefore ribonucleolytic [ 32 ] or biological activities [ 32 , 33 , 34 , 35 ] have been found in the recent past. Importantly, mutations affecting ANG ability to be translocated into the nucleus [ 36 ] have been detected in various cohorts of amyotrophic lateral sclerosis (ALS) and/or Parkinson’s disease (PD) patients [ 37 , 38 ].…”

“…Importantly, mutations affecting ANG ability to be translocated into the nucleus [ 36 ] have been detected in various cohorts of amyotrophic lateral sclerosis (ALS) and/or Parkinson’s disease (PD) patients [ 37 , 38 ]. In addition, we recently reported the effects of two ANG pathogenic variants on the tRNA processing and G-quadruplex formation [ 32 ].…”

“…Therefore, we studied here the self-association propensity of ANG and of three of its pathogenic variants found in amyotrophic lateral sclerosis (ALS) and Parkinson’s Disease (PD) patients, namely H13A-ANG, S28N-ANG, and R121C-ANG. We chose the first mutant because it is inactive, lacking one of the three catalytic residues [ 32 ]; the second had been envisaged in a previous report to be possibly inclined to dimerize [ 43 ], while the third displays a higher RNase activity than wt-ANG [ 35 ]. This has been ascribed to a decreased steric hindrance affecting the catalytic site cleft caused by the R > C mutation [ 35 ].…”

Tailoring the stress response of human skin cells by substantially limiting the nuclear localization of angiogenin

“…Conversely, if cells are subjected to a stress, the majority of ANG moves to the cytoplasm and is activated by dissociation from RNH1 to cleave tRNAs and release tiRNAs, actively involved in the cellular response to stress [ 15 , 29 ]. Interestingly, previous studies reported that also recombinant ANG, when added directly to culture media of multiple cell lines, is rapidly internalized and cleaves mature cytoplasmic tRNAs within anticodon loops [ 16 , 30 , 31 ]. This emphasizes even more the meaningfulness of dynamic subcellular localization of ANG in the cell and above all how cytoplasmic localization is the essential requirement for handling altered cellular homeostasis.…”