Human and Mouse Nephrin and Their Interactions With 13 Proteins: An In Silico Study

Background Human nephrin (hNeph) (podocyte protein) has been known to be involved in both the formation and maintenance of the slit diaphragm (SD) and also acts as a hub protein in the podocyte by modulating cell polarity, cell survival, cell adhesion, cytoskeletal organization, mechano-sensing, and SD turn-over. Methodology In the present investigation, we aimed to analyse the hNeph and mouse nephrin (mNeph) and their interactions with 13 proteins using the molecular docking method. The 13 selected human proteins which include matrix metalloproteinases (MMP 2 and 9), retinol-binding proteins (RBP 3 and 4), kallikrein 1 (KLK 1), uromodulin, insulin-like growth factor binding protein 7 (IGFBP7), cystatin C, podocin, beta arrestin 1, vang-like protein 2 (VANGL2), dynamin 1, and tensin-like C1 domain-containing phosphatase (TENC1) were studied on the docking analysis of hNeph and mNeph by using the HDOCK (protein-protein) docking method. In addition, the physicochemical (PC) properties of 15 proteins were performed using the ProtParam web server. Results In the present investigation, five chosen human proteins, namely, IGFBP7, cystatin C, podocin, VANGL2, and TENC1, have exhibited theoretical isoelectric point (PI) values greater than 7.0. The protein-protein docking analysis has shown that hKLK and hVANGL2 exhibited the maximum docking score of -206.39 kcal/mol and -329.28 (kcal/mol) with the target proteins mNeph and hNeph, respectively. Conclusions Thus, the current finding highlights the interactions of hNeph and mNeph with 13 chosen proteins, which may help in renal disease management.


Introduction
Nephrin is an essential component of the extracellular portion of the slit diaphragm (SD), which is situated between the basement form calyx of the glomerular filtration barrier and the foot process of podocytes along with endothelial cells.It is a transmembrane protein, with a molecular weight of approximately 180 kDa, and has 1241 amino acids [1][2][3].It plays a key role in the SD of podocytes, and it forms a molecular sieve that permits the passage of tiny molecules while blocking the loss of vital proteins and blood cells into the urine, hence enhancing the glomerular filtration barrier's selective permeability.However, in the case of hypertension, angiotensin II acts on the podocyte AT1-receptor to induce nephrin-β-arrestin2 binding and nephrin endocytosis, resulting in the enhancement of the glomerular permeability and in turn contributing to albuminuria in mice.The interaction between nephrin on podocytes and various other proteins, including those on endothelial cells and within the basement membrane, is critical for the proper functioning of the glomerular basement membrane (GBM).Disruption or dysfunction of nephrin can lead to various kidney diseases [4,5].With regard to diabetic nephropathy (DN), alterations in nephrin expression and its urinary excretion have been observed in the early stages.Numerous studies have reported significant increased urinary nephrin concentration with DN, particularly in microalbuminuria or overt proteinuria [6][7][8].Moreover, urinary nephrin levels have been correlated with the severity of renal histopathological changes and the progression of DN.Furthermore, urinary nephrin identification also plays a significant role in the early detection of severe pre-eclampsia [9].
Similarly, elevated urinary matrix metalloproteinase 9 (MMP 9) levels in DN patients have been reported [10].Hirata and colleagues have reported that the activation of the MMP 2 enzyme is involved in the pathogenesis of both hypertension and DN [11].Tsai and coworkers have demonstrated that the urinary retinol-binding protein 4 (RBP 4) levels have been increased in severe non-alcoholic fatty liver disease with both hypertension and proteinuria conditions [12].The other protein plasma kallikrein (KLK) has been reported to possess a protective effect on renal function especially in type 1 diabetes mellitus (DM) [13].Uromodulin has been reported as the principal urinary protein in healthy persons [14].Wang and colleagues have reported that the insulin-like growth factor binding protein 7 (IGFBP7) has been utilized as one of the biomarkers for the early diagnosis and prognosis of acute renal injury [15].Benoit and coworkers have reported the serum cystatin C as a biomarker for the early diagnosis of chronic renal disease [16].ElShaarawy and colleagues have demonstrated the urinary podocin as a novel biomarker for the early diagnosis of diabetic renal disease in type 2 DM [17].Xu and colleagues have reported that the beta arrestin 1 deficiency results in reduced renal fibrosis under the experimental mice model [18].Rocque and coworkers have demonstrated the physiological role of vang-like protein 2 (VANGL2) in the maintenance of glomerular structure as well as perm selectivity in the adult mice kidney [19].Khalil and colleagues have reported that the dynamin expression level has been elevated in human proteinuric kidney diseases [20].Lee and colleagues have reported that the tensin-like C1 domain-containing phosphatase (TENC1) has been known to be involved in the pathogenesis of diabetic kidney disease by inducing podocyte hypertrophy under hyperglycemic condition [21].
The interaction between nephrin on podocytes and various other proteins is important for renal physiological function, hence the present investigation where we selected 13 human proteins which include MMP 2 and 9, RBP 3 and 4, KLK 1, uromodulin, IGFBP7, cystatin C, podocin, beta arrestin 1, VANGL2, dynamin 1, and TENC1.These 13 human proteins were studied on the docking analysis of human nephrin (hNeph) and mouse nephrin (mNeph) by using the HDOCK (protein-protein) docking method, in order to know their interactions and binding affinities.

Preparation of target or receptor proteins (hNeph and mNeph)
The 3D structure of hNeph (AlphaFold DB: 060500) and mNeph (5ZYS.pdb)was downloaded from the Protein Data Bank (hNeph) and AlphaFold database (mNeph), respectively.A chain of hNeph and B chain of mNeph were prepared independently by carefully deleting other chains and heteroatoms including crystallographically observed water by applying the UCSF Chimera free software [22].

Docking study
The docking analysis was performed for 13 selected target ligands with that of hNeph and mNeph (target receptor proteins) individually using the protein-protein (HDOCK) method [24].
The HDOCK docking score results have exhibited the following order: hVANGL2 (-329.In the present study, there is no correlation between the PC properties and molecular docking analysis, since PC is an independent property.However, in the case of hNeph, the first four highest docking score proteins exhibited an instability index value greater than 40.Moreover, all the HDOCK docking scores of hNeph have exhibited more than -200 (kcal/mol) which might be due to larger molecular weight protein compared to that of mNeph.

Discussion
Nishibori and coworkers have reported that nephrin is confined to SD which is mainly based on its cytosolic interaction with podocin protein [25].Li and colleagues have reported that nephrin interacts with many other podocyte proteins as well as with SD protein and thus is involved in activating cell signaling pathways in podocytes [26].In vivo (mouse) knockout experimental models have reported that the impairment of nephrin (or) podocin (or) neph1 expression is enough to interrupt SD formation and stimulates severe disease within days and also even in utero [27].Nephrin interacts with beta arrestin, which in turn stimulates endocytosis in both clathrin-and dynamin-dependent modes of action [27].Coward and coworkers have demonstrated that nephrin is essential for the insulin action on glomerular podocytes of humans [28].Similarly, Soda and colleagues have reported that dynamin has been complexed with nephrin in an indirect way during its own endocytosis processes [29].Rocque and colleagues reported that the deficiency of VANGL2 activity which plays a vital role in the planar cell polarity (PCP) pathway leads to the elevated surface expression of nephrin in podocytes in mice (in vivo) experimental model [19].C1-Ten has been known as nephrin tyrosine phosphatase, which is reported to be up-regulated in DN and also involved in the stimulation of podocyte hypertrophy [21].Shree and coworkers have demonstrated the interaction of MMP 9 with nephrin using the protein-protein docking method [30].In the present study, seven selected proteins (hMMP 2, hMMP 9, hRBP 3, hRBP 4, hKLK, hUromodulin, and hBeta arrestin 1) have shown an instability index value lesser than 40 that demonstrate these said proteins are unstable in nature [24].Similarly, in the current investigation, all the chosen proteins (except hNeph) have exhibited the lowest grand average of hydropathicity of the protein (GRAVY) value which indicates these said proteins are known to have good interaction with water molecules [24].
With regard to the HDOCK (protein-protein) docking analysis, in the current investigation, all the docking scores have exhibited more than -200 kcal/mol with hNeph.On the other hand, all the docking scores have exhibited less than -200 kcal/mol (except for hMMP 9 and hKLK 1) with mNeph.In the present investigation, hMMP 9, with the following amino acid residues, namely, Tyr149, Gly186, Leu188, Ala189, His190, Ala191, Phe192, Pro193, Tyr218, His226, Gln227, His230, Asp235, His236, Pro246, Met247, and Tyr248, interacts with hNeph.These results were on par with earlier reports where Pro246 and Met247 amino acid residues of MMP 9 interact with nephrin using the ClusPro (protein-protein) docking method [30].To the best of our knowledge, no other proteins have been reported to interact with hNeph (or) mNeph so far.

Limitations and future recommendations
The current study findings are based on the HDOCK (protein-protein) docking analysis which provides new insight about these 13 proteins, namely, hMMP 2, hMMP 9, hRBP 3, hRBP 4, hKLK 1, hUromodulin, hIGFBP 7, hCystatin C, hPodocin, hBeta arrestin 1, hVANGL2, hDynamin 1, and hTENC1, as modular proteins of kidney function, and moreover, it is considered as a preliminary research work.Furthermore, in vitro (cell-based) and in vivo (mouse or rat animal model) experiments are needed to confirm these 13 ligands (proteins) as good modulating action against both hNeph and mNeph.

Conclusions
The present HDOCK (protein-protein) investigation has shown that all 13 proteins dock very effectively with the two target proteins, namely, hNeph and mNeph.Interestingly, in the case of hNeph, all the HDOCK docking scores have exhibited more than -200 (kcal/mol), which might be a larger molecular weight protein compared to that of mNeph.The protein-protein (HDOCK) docking analysis has shown that hVANGL2 exhibited the maximum docking score of -329.28 (kcal/mol) with the target protein hNeph.Thus, the current finding highlights the interactions of hNeph and mNeph with 13 chosen proteins, which may help in renal disease management.