Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. It is caused by selective destruction of motor neurons 
. A combination of several factors including protein aggregation, mitochondrial dysfunction, oxidative stress, defective axonal transport, excitotoxicity and dysfunctional growth factor signaling have been linked to the onset of ALS. In the later stages of the disease, paralysis sets in and death ensues due to respiratory failure. Approximately 10% of the cases are inherited in an autosomal dominant manner while 90% are sporadic. Among the catalogued ALS-like motor neuron diseases, mutations in SOD1
(ALS11) and a hexanucleotide-repeat expansion (GGGGCC
) in the C9ORF72 cause adult onset neurodegenerative disorder 
. Deletion of the hypoxia-response element in the vascular endothelial growth factor (VEGF
) promoter also caused adult onset motor neuron degeneration similar to ALS in mice 
. However, since mutations in VEGF
have not been found in ALS patients, VEGF
polymorphisms may be considered as a risk factor in some populations 
. Among the ALS types that portray the characteristic adult onset neurodegenerative disorders, about one-fifth of the familial cases are attributed to missense mutations in the gene that encodes SOD1. Mutations in SOD1
gene cause ALS through a toxic gain of function and not due to an impairment of its antioxidant function 
and hence SOD1
mimetics may not lead to an effective therapy. This and the fact that most reported cases of ALS are sporadic, underscores the importance of studying other gene mutations in detail.
Among other genes, heterozygous missense mutations in ANG
have been associated with ALS 
. ANG, a 123 amino acid single chain polypeptide (14.1 kDa), is strongly expressed in both endothelial cells and motor neurons in prenatal and adult spinal cord of humans. It influences the physiology and health of motor neurons by stimulating angiogenesis, neurite outgrowth and path-finding, and protects motor neurons under hypoxia 
. ANG maintains normal vasculature and thereby protects motor neurons from various stress conditions. Wu et al. 
have shown using angiogenesis, ribonucleolysis, and nuclear translocation assays that ANG mutations identified in ALS patients are associated with functional loss of angiogenic activity. Baker et al. 
and Cruts et al. 
have also observed null mutations in another angiogenic protein, progranulin (PGRN), in frontotemporal dementia (FTD) patients. Mutations of PGRN
gene were also reported in ALS patients 
. Since compromised angiogenic activity appears to play a pivotal role in ALS progression, a study of the effect of selected mutations on the function of ANG may help in defining a better therapy.
ANG executes its essential functions via three functional sites (). The first functional site, comprising the catalytic triad His13, Lys40 and His114, is responsible for ribonucleolytic activity. The second functional site consists of the nuclear localization signal 29
, which resides on the surface of ANG and facilitates its translocation into nucleolus. In endothelial cells and motor neurons, ANG undergoes nuclear translocation, binds to the promoter region of ribosomal DNA and helps in ribosome biogenesis, protein translation and cell proliferation by stimulating rRNA transcription. The third functional site is the receptor-binding site 60
, which is responsible for the binding of ANG to the endothelial cells, motor neurons and induces second messenger responses. Recent experimental studies have shown that mutations in ANG result in the loss of either ribonucleolytic activity, nuclear translocation activity or both and any single loss of either of these functions leads to the complete loss of angiogenic function which in turn causes ALS 
. More than 15 ANG
mutations have been associated with ALS of which 10 have been studied in detail 
. However, the molecular mechanism behind the functional loss of ANG due to these mutations is not completely understood.
Cartoon representation of X-ray structure for Human Angiogenin (PDB code: 1B1I).
In order to explain how these mutations resulted in the loss of either ribonucleolytic activity or nuclear translocation activity, or both, we have conducted a series of molecular dynamics (MD) simulations including all structurally different mutant forms that have complete ribonucleolytic and nuclear translocation activity information, except those near the catalytic site, so that our MD simulation results can be validated 
: (i) K17I which results in the loss of ribonucleolytic activity, (ii) S28N which has only 9% of ribonucleolytic activity and no nuclear translocation activity, and (iii) P112L which results in partial ribonucleolytic activity and complete loss of nuclear translocation activity. We also studied the V113I variant which is prevalent among Italian patients 
. In addition, our study included two missense SNPs, T195C and A238G at the gene level encoding L35P and K60E mutants respectively, not yet clinically correlated with ALS 
. We performed 50 ns duration MD simulations of the WT-ANG, disease associated K17I, S28N, P112L and V113I variants and L35P and K60E mutants with AMBER 10 software suite. The sites of these mutations are shown in .
Ribbon representation of mutational sites in Human Angiogenin.
This is the first study using MD simulations that presents an explanation for the loss of functions observed in ANG mutations. Our MD simulations demonstrate that a possible molecular mechanism may involve a change in conformation of the catalytic triad residue His114 resulting in the loss of ribonucleolytic activity. Simulation results confirm structural instability of ANG variants as reported in experimental studies. Further, we predict that L35P mutant may be responsible for causing ALS by loss of ribonucleolytic activity as well as nuclear translocation activity while K60E may be passive.