PITRM1 interaction studies with amyloidogenic nonapeptide mutants of familial Alzheimer’s disease

Abstract Amyloid β-protein (ABP) is found to be the major cause for the development of neurodegeneration which leads to Alzheimer’s. The Aβ nonapeptide segment, QKLVFFAED (amino acids 15–23) is the highly amyloidogenic central region of Aβ. Familial mutation in Aβ increases the aggregation property of the peptide compared to the Native (Wild) amyloid-beta (Aβ) and these mutations fall on the Aβ nonapeptide segment. The catalytic activity of pitrilysin metallopeptidase 1(PITRM1) with familial mutant Aβ (Flemish, Arctic, Dutch, Italian and Iowa) during interaction is examined using molecular dynamic simulation. The molecular dynamics simulation of PITRM1 and the Aβ nonapeptide segment showed similar RMSD with respect to stability. The active site amino acid (AA) H108, hydrophobic pocket AA residues L111, F123, F124, and L127 and the basic pocket AA residues R888 and H896 showed similar interactions with both wild and familial Aβ. The molecular level interaction between amyloid beta and PITRM1 were similar in the wild and familial mutants except for the Arctic mutant. The hydrophobic interaction was commonly observed between the S1 hydrophobic pocket and the LVFF region, the Arctic mutant showed less hydrogen bond formation consistently when compared to other complexes. This molecular information on catalytic activity suggests that modulating inactive PITRM1 or an increase in expression of PITRM1 can help in eliminating different kinds of familial mutant Aβ in neurodegenerative cells. Communicated by Ramaswamy H. Sarma


Introduction
Amyloid b-protein (ABP) is considered to be the leading cause of the neuropathological condition in Alzheimer's disease (AD). Ab pays the way for neuronal damage leading to Alzheimer's condition. Initially, Ab is derived from the sequential cleavage of amyloid precursor protein (APP), followed by deposition and aggregation (Serrano-Pozo et al., 2011). The sequential cleavage of the peptide is key to the formation of plague. a-secretase, b-secretase and are involved in the sequential cleavage of the APP at residues Lys687/Leu688, Met671/Asp672 and Val711/Ile712 or Ala713/ Thr714, respectively (Serpell, 2000;Serrano-Pozo et al., 2011). Influence in the amyloid-beta (Ab) degradation pathways can induce the accumulation of peptides and lead to neuronal toxicity and neuronal death.
The rate of aggregation by Ab is affected by the change or mutation in single amino acids. In familial mutation, the aggregation property of the peptide is highly enhanced (Kim & Hecht, 2008). There are over 50 different mutations found in APP which lead to the early and late onset of the neurodegenerative condition (Janssen et al., 2003). In particular, certain mutations are produced in the single amino acid of the Ab which can cause a familial form of Alzheimer's disease. Familial AD (FAD) is associated with the mutations in presenilins 1 and 2 and the amyloid precursor protein (APP) (Weggen & Beher, 2012). These include specific mutation at residue Flemish (A21G) (Hendriks et al., 1992), Arctic (E22G) (Nilsberth et al., 2001), Dutch (E22Q) (Fernandez-Madrid et al., 1991;Levy et al., 1990;Van Broeckhoven et al., 1990), Italian (E22K) (Bugiani et al., 2010), and Iowa (D23N) (Grabowski et al., 2001) disease forms. Ab FAD mutations, such as the Italian (E22K) and Arctic (E22G) mutations, are believed to exert their pathogenic effects by inducing the formation of stable oligomers and protofibrils (Masuda et al., 2008;Nilsberth et al., 2001). Flemish (A21G) and Arctic (E22G) mutations have been reported to lead to the adoption of distinct aggregate structures, as determined by ion-mobilitybased mass spectrometry (Gessel et al., 2012). The A21G mutation is unique among the CAA-causing FAD mutations and it increases the total Ab production causing biochemical and structural alterations in Ab (Hatami et al., 2017;Van Nostrand et al., 2001). Thus far familial mutation has also been a major cause in the pathology of AD progression.
The distribution of Ab is also observed in organelles like mitochondria, Endoplasmic reticulum, Golgi complex, endosomes, lysosomes, multivesicular bodies and cytosol (LaFerla et al., 2007). The early intracellular accumulation of Ab is also a reason for neuronal stress leading to neuronal death. Accumulation of Ab 1-42 in the intracellular organelles is considered recently because of the relation between accumulation and oxidative stress caused during aggregation (Lustbader et al., 2004). Mitochondria is one of the organelles in which the Ab plague has been observed in the membrane causing structural and functional damage leading to neuronal cell death (Reddy, 2009). The presence of Ab in mitochondria has been characterized recently and many peptidase enzymes also interact with the same to degrade (Falkevall et al., 2006;Hansson Petersen et al., 2008;Manczak et al., 2006). Accumulation of the Ab inside mitochondria induces ROS production thereby leading to neuronal cell death (Cha et al., 2012;Chen & Yan, 2010). There are Ab degrading enzymes like neprilysin, insulin-degrading enzyme, angiotensin degrading enzyme and human endothelin degrading enzyme (Miners et al., 2011). The degradation ability of these proteases plays a major role in reducing the Ab concentration in the AD patient's brain. Understanding the physiological condition and performing structural studies for the specificity of Ab can help in designing an efficient modulator for the Ab degrading enzymes. These modulators can in turn help in providing an increased and prolonged activity of the peptidase and result in the decrease of Ab peptide. In this respect, we have focused on the proteolytic mechanism of PITRM1 in Ab-peptide degradation across different mutant Ab peptides like Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N).
Pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme that degrades the transit peptides and the mitochondrial fraction of Ab inside mitochondria (Hansson Petersen et al., 2008;Pagani & Eckert, 2011;Pinho et al., 2014;Stahl et al., 2002). The protein is 114 kDa with a large 13,300-A chamber for substrate interaction and cleavage (King et al., 2014). PITRM1 has 3 region N region (33-509), helical hairpin region HP (510-575) and C region (576-1037). They can accommodate approximately 65 residue peptides inside the large chamber and can degrade a peptide as small as leu-enkephalin (Chow et al., 2009). The N region of PITRM1 is the peptide binding site which has two hydrophobic pockets near the active site (L111, F123, F124 and L127). PITRM1 resembles the Insulin degrading enzyme (IDE) which is a Pitrilysin family but it cannot degrade insulin. In the absence of exosite in the catalytic chamber, PITRM1 cannot degrade insulin but has an affinity toward Ab peptide. This makes PITRM1 an important target for the clearance of Ab in AD pathogenesis. A decrease in PITRM1 activity can lead to Ab aggregation and neuronal cell death. An increase in ROS affects the degrading capacity of PITRM1, similarly, the activity decreases with age in AD affected brain. Suggesting that wild Ab itself becomes resistant to cleavability by PITRM1 over time due to factors like ROS. These indicate that PITRM1 play an important role in Ab degradation and can reduce the concentration of Ab in the AD brain.
Thus, familial mutations in the Ab nonapeptide play a major role in aggregation and plaque formation. Mutational can hinder the cleavability of the peptide by peptidases. Since these mutant peptides are comparatively aggregation-prone than the wild type. It is important to understand the availability of these mutant peptides to interact with the PITRM1 and undergo degradation. Understanding the physiological condition and performing structural studies for the specificity to Ab can help in designing an efficient modulator for the Ab degrading peptidase enzymes. Hence the present study is to understand the behaviour and response of peptidases during the degradation Ab nonapeptide region using molecular docking and molecular dynamic simulation studies. Thus, this study will give a brief insight on the substrate Ab recognition and degradation by PITRM1 to develop a potential modulator of PITRM1 and use it in future treatment against AD.

Data mining and refined 3D model building of PITRM1
The molecular docking studies were performed using the modelled protein structure of PITRM1. Swiss model workspace was used to develop the 3D structure of PITRM1 using the template 4NGE chain A (Arnold et al., 2006;Biasini et al., 2014) and further energy minimization for the structure was performed using Swiss-PdbViewer (SPDBV) (Kaplan & Littlejohn, 2001). The Ab peptides for docking studies were prepared by using 4NGE chain B as a template and mutating the nonapeptide sequence QKLVFFAED at A21G, E22G, E22Q, E22K and D23N followed by energy minimisation using SPDBV.

Preliminary docking of PITRM1 and familial mutant ab peptide
The interaction between the familial mutant Ab nonapeptide (5 types), Wild Ab and PITRM1 was carried out using online servers GRAMM-X (Tovchigrechko & Vakser, 2006). GRAMM-X employs Fast Fourier Transformation methodology, smoothed potentials, refinement stage, and knowledge-based scoring. The modelled protein and peptide are uploaded and potential receptor interface residues were selected for docking. The Docked structures were refined using FiberDock (Mashiach et al., 2010), an efficient method for flexible refinement and rescoring of rigid-body protein-protein docking.

Molecular dynamic simulation
Molecular dynamic simulations of the PITRM1 familial Ab mutant protein-peptide complex were performed using the GROMACS 5.1.2 package for 100 ns (Van Der Spoel et al., 2005). The protein-peptide complex structure was solvated in a triclinic water box under periodic boundary conditions using a 0.9 nm minimum distance from the protein to the box faces and neutralizing the system using two Clions added to the solvent. The final system consisted of approximately 25,000 atoms. Following the steepest descent minimization, the systems were equilibrated in the canonical ensemble (under NVT conditions for 500 ps at 300 K) and, subsequently, in the isothermal-isobaric ensemble (under NPT conditions for 500 ps) by applying position restraints to the protein. Lastly, all the restraints were removed, and 30 ns molecular dynamic runs were performed twice under NPT conditions at 300 K. To maintain constant pressure (1 atm), (isotropic coordinates scaling), the Parrinello-Rahmanbarostat was used with a relaxation time of 2.0 ps. Van der Waals interactions were modelled using 6-12 Lennard-Jones potentials, with a 1.4 nm cut-off. The long-range electrostatic interactions were calculated using the PME method, with a cut-off for the real space term of 0.9 nm. Covalent bonds were constrained using the LINCS algorithm. The time step employed was 2 fs, and the coordinates were saved every 2 ps for analysis, which was performed using standard GROMACS tools (Kumar et al., 2021). MM/PBSA energies were calculated on the last 10 ns using the tool g_mmpbsa for free energy calculation between PITRM1 and the Ab nonapeptide (Kumari et al., 2014). Further, the trajectory analysis was carried out by calculating the RMSD (Root mean square deviation), RMSF (Root mean square fluctuation), Gyration, Hydrogen bond and the minimum distance between peptidase and Ab nonapeptide and plotted in the Grace plotting tool. Pycontact is used to analyse the protein peptide interaction for the 100 ns simulation output of GROMACS (Scheurer et al., 2018). The results are expressed as the number of hydrogen bonds (contact score). PLIP was used to study the molecular interaction between the protein peptide for the average structure from molecular simulation (Salentin et al., 2015).

Molecular modelling and docking
The 3D model structure of PITRM1 was modelled using the Swiss model server. The best model was selected based on the GMQE and Q mean values of 0.98 and 0.07 respectively. The wild-type Ab sequence was retrieved from 4NGE chain B and used to develop the familial mutant Ab sequences using the mutation option in SPDBV. Five different mutants Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N) were developed and followed by energy minimization (Figure 1). Docking studies using GRAMM-X demonstrated the protein-peptide interaction/docking pose similar to that of the crystal structure 4NGE, further the affinity was calculated using the FiberDock (Table 1). The global energy was calculated showing mutants have better interaction when compared to the wild type.

Molecular dynamic simulation of Ab and PITRM1
Molecular dynamics simulation can give a brief idea about the molecular level effect of mutation during the interaction. Ab nonapeptide (QKLVFFAED) is an amyloidogenic region that is prone to form aggregation. The shortest core fragment critical to fibril formation lies in the Ab (16 À 22) region, which also has the hydrophobic core residues LVFF. The KLVFFAE, region is well enough to initiate aggregation seed. The effect of familial Ab mutations and their interaction with PITRM1 is studied and the structural dynamics and energetic effects of the familial mutation are examined. MD simulation was performed between the five familial mutants (A21G, E22G, E22Q, E22K and D23N)/ one Wild Ab nonapeptide and PITRM1 protein for a period of 100 ns using GROMACS. The objective of the study focuses on understanding the interaction between familial mutants and PITRM1. The calculation was carried out in the forms of PITRM1-Ab nonapeptide complex, PITRM1 protein alone, N region of PITRM1, HP region of PITRM1, C region of PITRM1 and the Ab nonapeptide.
3.3. Stability of the PITRM1 and Ab complex (Wild (native), Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N)) The RMSD and RMSF calculation along the time of simulation allows for checking the stability and structural equilibrium of the protein-peptide complex based on structural changes. The root mean square deviation of the six simulated complexes was calculated. The overall RMSD between the six complexes were more are less similar that the stability of the complex reached within 2 ns of the simulation. The last 50 ns (between 50 ns and 100 ns) were used for the analysis of the RMSD ( Figure 2). The PITRM1 showed an average RMSD of 5.75±0.2nm, 5.54 ±0.16 nm, 4.8 ±0.15 nm, 4.71 ±0.32 nm, 5.43 ±0.13 nm and 5.11 ±0.38 nm for Wild, Flemish (A21G), Italian (E22K), Dutch (E22Q), Arctic (E22G), and Iowa (D23N), respectively ( Figure 2). The N region showed an average RMSD of 4.31±0.24nm, 4.60 ±0.12 nm, 4.13 ±0.17 nm, 4.06 ±0.14 nm, 4.56 ±0.11 and 4.16 ±0.19 for Wild, Flemish (A21G), Italian (E22K), Dutch (E22Q), Arctic (E22G), and Iowa (D23N) respectively (supplementary material Figure S1A). The RMSD of the PITRM1-HP region which contains the hairpin loop showed the highest RMSD in the whole protein with 7.38 ±0.48 nm for wild type, 8.03 ±0.30 nm for Flemish, 6.04 ±0.31 nm for Italian, 5.03 ±0.32 nm for Dutch (E22Q), 7.44 ±0.24 nm for the Arctic and 6.89 ±0.41 nm for Iowa (D23N) (supplementary material Figure S1B). The C region of PITRM1 showed a higher average RMSD when compared to the wild (5.06 ±0.13 nm). An RMSD of 5.00 ±0.26 nm,4.54 ±0.22 nm, 4.47 ±0.32 nm,4.47 ±0.11 nm and 4.61 ±0.25 nm for Flemish (A21G), Italian (E22K), Dutch (E22Q), Arctic (E22G), and Iowa (D23N)were observed respectively (supplementary material Figure  S1C). The RMSD of Ab nonapeptides was in the range of 0.7 and 0.4 for all the peptides both Wild and familial mutant (supplementary material Figure S1D). The RMSD of the complex and PITRM1 alone remained to be same for all the wild and familial mutant Ab interaction simulation models. The analysis based on the individual region like C, HP and N regions of PITRM1 there was a change in C and N regions, but RMSD was observed high in the HP region (supplementary material Figure S1). PITRM1 has three region N region (33-509), helical hairpin region HP (510-575) and C region (576-1037) (Figure 3(A)). PITRM1 chamber consists of two pockets that act as an active site aminoacid H108. The first hydrophobic pocket consists of amino acids L111, F123, F124, and L127 and the second, basic pocket is comprised of amino acids R888 and H896. The amino acid between 510-575 for a hairpin loop region (HP) is between the N and C region of PITRM1 and the loop region. The active site amino acid H108 showed a fluctuation of 0.0827 for wild and 0.1524, 0.1235, 0.1369, 0.1503 and 0.1998 nm for Flemish, Italian, Dutch, Arctic and Iowa respectively ( Table 2). The RMSF analysis of the first hydrophobic pockets of the Iowa, Dutch and showed higher fluctuation when compared to wild type. Flemish and Italian in the region showed fluctuation similar to that of wild type. The second basic pocket also showed higher RMSF when compared to native in the order of Iowa, Italian, Arctic, Dutch and Flemish. The major fluctuations were observed in the HP hinge region and the loop region of PITRM1 in all complexes (Figure 3(B-G), supplementary material Figure  S2A-C). The free-moving region facilitates the accommodation of Ab peptides inside the cavity region. Accommodation of large peptides like Ab can be challenging, the hydrophobic exosite region L348, L428, I432, F344 and L465, L60, F443, L447 and Y450 facilitates the accommodation of the peptide (Table 2). L60 and L447 showed major fluctuations in all the types of the peptide during the simulation.

Discussion
Mutation in Ab directly affects the pattern of aggregation, leading to the difference in the availability of peptides for degradation (Chen et al., 2012;Chong et al., 2013;Kim & Hecht, 2008;Qin & Jia, 2008). The effect of the mutation in   Ab can also affect the degradability when a protease act on it. The familial mutant and the wild type both are prone to aggregation and the difference relay on how efficient and faster these peptides are been degraded by the peptidases (Kim et al., 2007;P erez et al., 2000). PITRM1 is one such protein that degrades the Ab, these peptidases cut the peptide based on the mutations studied here including Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N) along with the wild Ab nonapeptide. Insulin degrading enzyme a related family member of PITRM1 is known to degrade Ab. The ability to degrade the other forms of mutant peptides is also observed in IDE. The difference in affinity due to mutations has made Ab resistance/less prone to the degradation process. Understanding the reason for this will help in designing a small molecule/peptide for enhancing protease activity thereby helping in the treatment of AD.
In the present study, the mutation in the Ab (familial mutation) was created in a Ab nonapeptide QKLVFFAED and docked with the protease PITRM1. The docked complex was subjected to simulation studies to understand the molecular interaction between them. The RMSD, RMSF and Rg of the PITRM1-ab complex were calculated against the initial structure. The interaction of the PITRM1 and Native (Wild) started to stabilize after 5000ps which was seen in the RMSD similarly to the PITRM1-Nonapeptide A21G mutant, PITRM1-Nonapeptide E22G mutant, PITRM1-Nonapeptide E22Q mutant, PITRM1-Nonapeptide E22K mutant and PITRM1-Nonapeptide D23N mutant which also started to stabilize after 5-10 ns. This stabilisation process of the peptidase Ab complex within the initial 2 ns is also observed in studies with Insulin degrading enzyme Ab simulation studies (Bora & Prabhakar, 2010).
The RMSD were in the range of 0.5 to 0.6 nm for wild and mutant abs in the last 50 ns MD period. The RMSD were comparatively lower than the wild type in the last 50 ns of simulation. The accommodation of peptides into the cavity plays an important role in the degradation of the peptides. The wild type peptide showed higher RMSD when compared to all other Ab nonapeptides. The ability to protofibrilation Figure 7. Image showing Pycontact interaction between PITRM1 and amyloid beta for 50 ns (last 50 ns of 100 ns simulation). X axis represents the amyloid beta nona peptide (wild and familial types) and the y axis represents the amino acids of PITRM1 involved in the interaction. Hydrogen bond (blue), Hydrophobic interaction (orange), others (green) and salt bridges (red) are the different types of bonds formed respectively. depends on the region 15-23 of Ab, which also contribute to faster protofibrilation. The familial mutants Arctic (Nilsberth et al., 2001), Dutch and Iowa  has an increased rate of aggregation when compared to the wild type. The availability of these familial mutant peptides gets reduced in Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N) from the RMSD observations. PITRM1 reaches stability during the interaction with Ab nonapeptide at a different rate when compared between the familial mutants. Arctic and Iowa average RMSD were low compared to the wild and other familial mutants as it can form stable folding faster during the interaction with the fibrillogenic region. These Arctic and Iowa were identified to be exhibiting higher fluorescence while performing the Thioflavin fluorescence aggregation assay (Hatami et al., 2017). The Arctic mutation was one of several pathogenic APP mutations found to confer resistance to neprilysin-catalyzed proteolysis of Ab40 (Tsubuki et al., 2003). The HP region of the protein showed higher RMSD as the hinge region is set, so the C-region and N-region facilitates interaction with Ab nonapeptide. The minimum distance for interaction with the Ab nonapeptide and PITRM1 was maintained for the Wild and familial mutants. The N region is composed of a conserved region and uniformly negatively charged to facilitate interaction with Ab (Hatami et al., 2017). The C region lacks a positively charged surface that is essential for degrading intact insulin (McCord et al., 2013), showing the Ab nonapeptide had a larger interaction space between them. This facilitates the binding and accommodation maintaining very less distance during the interaction. Similarly, the IDE chamber is required to stabilize the catalytic cleft, the cleavage of these peptides occurs stochastically at a few predefined sites, and cleavage proceeds progressively (Guo et al., 2010;Lai et al., 2018;Manolopoulou et al., 2009).
In the familial mutant Ab the amino acids are Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E22K), and Iowa (D23N) Artic mutant showed a high fluctuation when compared with wildtype. The hydrophobic pocket of PITRM1 adjacent to the active site includes the amino acids L111, F123, F124 and L127 (King et al., 2014), the fluctuation in these amino acids was very high while interaction with E22G mutant followed by A21G and E22G mutants. These amino acids are highly conserved hydrophobic S1 sites. The E22K and D23N mutants showed fewer fluctuations when compared to the wild type. The major fluctuation was due to the fibrillogenic region forming the aggregation in the E22G variant. The amino acids R888 and R896 contribute to the second basic pocket of PITRM1 and were also involved in maintaining the stable structure. The overall dimension of protein due to the secondary structure's compact packing was analysed based on the radius of gyration. The compactness between the wild type and the mutant peptides showed no difference. The presence of a long dual Harpin switch c region and a larger surface between the N and C region of PITRM1 which operates as a 'book-opening' mechanism to accommodate the peptide makes the protein to be compact during the interaction. Molecular interaction of PITRM1 active site (H108) was observed with F20 wild type Ab Ab nonapeptide, similar interaction was also observed for Flemish (A21G), Arctic (E22G), and Iowa (D23N). The hydrophobic S1 region adjacent to the PITRM1 active site consists of bulky hydrophobic residues (L111, F123, F124 and L127) which interact with the hydrophobic residues in the Ab nonapeptide region (L17, V18, F19, F20, A21). The interaction of the PITRM1 involves the 4 amino acids in the S1 site with the F19, F20 and A21 wild type Ab nonapeptide. Flemish (A21G), Arctic (E22G), and Iowa (D23N) also showed a similar type of interaction to that of wild type hydrophobic interaction. Italian (E22K) and Dutch (E22Q) showed comparatively lower hydrophobic interaction. The presence of hydrophobic residues in the S1 site creates a unique region for its substrate preference during the degradation process. The Flemish (A21G) mutation has both the wild and mutant types to be aliphatic amino acids, as a result of which the changes considering the RMSD, RMSF, gyration and hydrogen bonds were minimal. The amino acid at the 21st position of Ab nona peptide does not influence the interaction process. The hydrophobic interactions were observed between the S1 hydrophobic pocket and the LVFF region of the Ab nonapeptide. The hydrogen bond formation and other interactions facilitated the higher free energy compared to wild because of the bond formation in amino acids at 15 and 16 for wild type whereas Flemish showed hydrogen bond at the 15,16 and 20th amino acids. The presence of lysine, an aliphatic amino acid in Italian (E22K) facilitates the hydrophobic interaction at the 22nd position which was absent in wild type. The free energy calculation also implied high binding energy compared to the other mutants and wild types. Italian and Iowa were observed to have similar free energy and the hydrophobic interaction were also similar but with a difference in electrostatic energy and Van der Waal forces. The presence of glycine in the 23rd position of the Arctic mutant made the complex to be less interactive due to its small structure. There were less hydrogen bonds and the hydrophobic  interaction maintained in the system. The system showed high electrostatic energy and high polar solvation energy. Even though the simulation results imply the ability of PITRM1 to interact with the respective familial mutants, the aggregation of Ab can be a key factor. The availability of peptides for the degradation during the process of neurodegeneration and the ability to enhance the activity will be vital in future studies.

Conclusions
Alzheimer's disease is one of the elderly neurodegenerative disorders which has been due to the specific nature of Ab aggregation in the neuronal cells. These aggregations are prone due to the region QKLVFFAED which is essential for plaque seed formation. This in turn induces further aggregation of protein leading to oxidative stress in neuronal cells leading to cell death. Various strategies have been adopted to reduce protein aggregation directly and indirectly.
Degrading the Ab peptide is the direct way in which the peptide can be digested by a peptidase. There are different peptidases, PITRM1 is a mitochondrial peptidase that can degrade Ab. The present study focuses on the ability of PITRM1 to degrade Ab, the question that arises is whether using PITRM1 can the same be applicable for the other familial mutant Ab. The familial mutants are aggregation-prone more than the wild ab, hence a target must be been chosen so it can be used as a versatile one. From this study, it is evident that, understanding the interaction between PITRM1 and familial mutants in developing modulators for PITRM1 in future.