Mechanistic Study On The Function Rescue Of SAMHD1 N-Terminal Fragment

SAMHD1(SAM and HD domain containing protein 1)is the first d NTP hydrolase found in human cells,and is an important immune regular and antiviral factor in the innate immune system.Since first reported in 2000,SAMHD1 has been associated with cell cycle regulation,cancer occurrence and development,viral infection suppression.Currently,it is widely accepted that,the C-terminus is the key part to perform all known functions of SAMHD1;on the contrary,no known function has been linked with the N-terminal fragment,except for existence of a nucleus localization signal.To examine the impact of N-terminal fragment upon SAMHD1’s functions,we conducted serial experiments and found that:1.The existence of an N-terminal fragment grants SAMDH1 the ability to suppress LINE-1 retrotranspositionSAMHD1 effectively suppresses the replication of LINE-1 retroelements,by which SAMHD1 regulates the activation levels of the innate immune system and represses the occurrence and development of cancer.Thus,it would be necessary to further explore the mechanism of SAMHD1-mediated LINE-1 suppression.Unexpectedly however,while confirming the reported mechanism on SAMHD1 inhibiting LINE-1,we found that tagging the N-terminus of SAMHD1 120,which is unable to suppress LINE-1,could rescue its potency against LINE-1 replication.Addition tests indicated that,N-m Cherry-SAMHD1 120 effectively reduces the protein levels of LINE-1 ORF2 p,which is similar to the mechanism how wild type SAMHD1 regulates LINE-1 activity.Notably,the amino acid sequences of m Cherry and the original N-terminal fragment of SAMHD1 are significantly different,suggesting that the existence of an N-terminal fragment,but not its specific sequence,might be necessary for SAMHD1 to perform its functions,at least some of them.2.The existence of an N-terminal fragment determines whether SAMDH1 could be depleted by VpxVpx-induced SAMHD1 depletion through proteasome-mediated proteolysis is the major pathway how HIV-2 and SIV counteract the antiviral ability of SAMHD1,while SAMHD1 120 could no longer be reduced by Vpx.To further verify the hypothesis that the existence of an N-terminal fragment is necessary for SAMHD1’s functions,we in this study examined the depletion of N-m Cherry-SAMHD1 120 in the presence of Vpx.As a result,both N-m Cherry-SAMHD1 120 and N-EGFP-SAMHD1 120,which not only indicated that the existence of an N-terminal fragment is critical for SAMHD1 to be degraded by Vpx,but also confirmed that such a phenomenon requires no specificity on the sequence of the N-terminal fragment of SAMHD1.Further tests showed that,both SAMDH1 120 and N-m Cherry-SAMHD1120 can be recognized by Vpx,while only N-m Cherry-SAMHD1 120 could be polyubiquitinated.All these data indicated that,during the process of Vpx-induced SAMHD1 degradation,the N-terminal fragment of SAMHD1 most likely shortens the distance to the ubiquitin ligase through spatial extension,and provides amino acid residue(s)essential for ubiquitination.In general,the N-terminal fragment of SAMHD1 is important for the protein to suppress LINE-1 retrotransposition and to be depleted by Vpx.Such an importance comes from the existence of the fragment itself,but not from the sequence specificity,suggesting that the spatial occupation might be critical for SAMHD1 to perform part,if not all,of its functions.Our data and future studies on such a topic would help people to understand the mechanisms of SAMHD1’s functions.On the other hand,a block in space would in principal sabotage Vpx’s ability of SAMHD1 depletion,providing novel theory and targets for the development of new drugs on AIDS treatment.