In questo studio porto dei dati a favore di un incremento dell’attività proteolitica all’interno delle NK, nei pazienti CFS. In particolare ipotizzo che la ridotta citotossicità delle NK sia dovuta alla digestione della perforina citoplasmatica, riconducibile a qualche enzima proteolitico, secondo una dinamica simile a quella descritta alcuni anni fa per l’Rnase-L.
Rnase-L and perforin are two proteins involved in host defense against viral infections. In chronic fatigue syndrome (ME/CFS), a disabling pathology of unknown etiology, the pathway which leads to the activation of Rnase-L and the genetic expression of NK cells perforin have been reported to be both up-regulated. Moreover, while researchers have described the presence of truncated forms of Rnase-L in ME/CFS patients, another work documents a depleted intracellular perforin level in NK cells from ME/CFS subjects. In this paper, I propose the hypothesis that the same proteolytic activity is involved in both Rnase-L and NK perforin cleavage. This idea is based on the identification of a significant local alignment between their two primary structures, which involves sub sequences that are surface exposed in both tertiary structures. This peptide divides Rnase-L into two truncated forms, one of which with the same length as the one described in ME/CFS patients. Moreover, it represents a possible B cell epitope for human perforin and this might provide an explanation for the low level of intracellular perforin reported in NK cells from CFS patients, as discussed below.
As a consequence of a viral threat, our own cells which are infected by pathogens start releasing INF-α, a cytokine which activates natural killer cells (NK), main actors of the innate immune system anti-viral response (Sompayrac, 2012). INF-α has other functions as well, and one of them is to promote the synthesis of the active form of Rnase-L, which is an enzyme implicated in the degradation of viral RNA (Bastide, et al., 2002). Rnase-L is a protein 741 amino acids long, with a mass of 83,533 kDa (ID: Q05823 ↑). It has been demonstrated that in ME/CFS a novel form of this molecule is present, with a weight of only 37 kDa, which is considered to be a dysfunctional variant of the 83 kDa peptide (Suhadolnik, et al., 1997)↑. It accumulates in peripheral blood mononuclear cell (PBMC) extracts from ME/CFS patients and has been considered a potential diagnostic marker (De Meirleir, et al., 2000)↑. Some years later, an increased proteolytic activity in PBMC extracts from CFS patients, has been demonstrated to be responsible for the accumulation of the truncated form. More precisely, researchers found that proteases in CFS PBMC extract, as well as purified human leukocyte elastase, give rise to two fragments of Rnase-L, the one already found in previous studies and another one of 30kDa. The 37 kDa fragment includes the N-terminal end of the full size Rnase-L, while the 30 kDa fragment includes the catalytic site in the C-terminal part (Demettre, et al., 2002)↑. Moreover, it was previously found that the whole pathway which leads to Rnase-L activation is up-regulated in ME/CFS patients with low molecular weight Rnase-L (Suhadolnik, et al., 1994)↑.
Perforin (ID: P14222 ↑) is an enzyme of 555 amino acids, with a weight of 61,7 kDa, used by NK cells and T CD8+ cells to induce apoptosis of infected cells, a process called cytotoxicity. More in detail, perforin forms pore-like structures to facilitate the entry of granzymes into the target cell, and granzymes activate several apoptosis pathways that ensure effective killing of the target cell (Sompayrac, 2012). It has long been recognized a cytotoxicity defect in NK cells from CFS patients, since first attempts in understanding the immunology of ME/CFS (Caligiuri, et al., 1987)↑. It was subsequently possible to demonstrate that low cytotoxicity of NK cells from ME/CFS patients is associated with a low level of perforin within NK cells (Maher, et al., 2005)↑. Surprisingly, a higher than normal perforin gene expression was found some years later, despite both the low NK cytotoxic activity and intracellular level of perforin in ME/CFS patients. In fact, in a 2011 study, the level of perforin mRNA was found to be elevated in NK cells from CFS patients (Brenu, et al., 2011)↑.
You would have noticed that both intracellular NK perforin and Rnase-L share the same pattern of a depleted level and an up-regulated pathway, in ME/CFS patients. In the case of Rnase-L, it has been possible, as mentioned, to further specify that low level of full length enzyme is due to the presence of a low molecular weight Rnase-L. I propose the hypothesis that perforin shares the same fate as Rnase-L of being digested by some proteases within NK cells, in ME/CFS patients. In order to legitimate this hypothesis, I am going to demonstrate the presence of a local similarity between primary structures of Rnase-L and perforin. I will show then that this shared peptide is a possible target for proteolytic activity as it is surface exposed in both these molecules. Moreover, I will provide a possible explanation for the finding of a low level of perforin in NK cells from MECFS patients.
In search for similarities between Rnase-L and perforin
Now, we will perform a local alignment between human perforin (ID: P14222 ↑) and Rnase-L (ID: Q05823 ↑) in search for some common peptides between the two primary structures through SIBI’s LALIGN (↑), using the following settings (default settings):
Remember that LALIGN uses a gap model op_gap + ex_gap(x), where x is the length of the gap; thus an opening gap of -12 with an extending gap of -2 corresponds to an opening gap of -14 with an extending gap of -2 in a gap model of the type op_gap + ex_gap(x-1). LALIGN gives us the following best alignment:
For settings in Eq. 1, LALIGN calculates a value of 0.1887 for λ, and of 0.02798 for K. Thus, according to local alignment statistic (Karlin, et al., 1990)↑ we have that the number p of local alignments between two random sequences of 741 and 555 amino acids, with a score at least S, is
Consider now that the score is calculated according to BLOSUM50, with a scale ln(2)/3. In order to calculate the score S to put in Eq. 2, we have to change the scale of the scoring matrix:
According to Eq. 2, the number p of local alignments with a score at least 61.22 is
A local alignment is usually considered to have good significance when it reaches a p of 0.05 (Altschul, 1991)↑, nevertheless two times that threshold value could still be of interest.
The 30kDa Rnase-L fragment
Demettre and colleagues were able to identify that the N-terminal end of the 30kDa Rnase-L fragment was 500-HLADFDKSI-508 in case of digestion by human elastase, and 492-LIDSKKAAH-500 when cleavage was obtained through PBMC extract from CFS patients (Demettre, et al., 2002)↑. Interesting enough, in both cases we have a fragment which may be produced when the target of the proteolytic activity is the local alignment found for Rnase-L and perforin. A way to further verify this hypothesis is to see if the peptide found in the local alignment is surface exposed and thus available to proteolytic enzymes. Rnase-L structure has been experimentally determined by X-ray diffraction method, with a resolution of 2.31 Å (Han, et al., 2014)↑. Its ID in the molecular modelling database (MMDB) is 118306, while its protein data bank (PDB) ID is 4OAU. You can find molecule’s 3D structure in this page (↑). I have downloaded its data in ASN.1 format and I have read it with Cn3D 4.3.1 (↑) and then I have searched for the peptide of the local alignment indicated above (Table 1) and highlighted it in yellow. As you can see (Figure 1) this peptide is surface exposed in its central residues, even if it has not a good protrusion from the molecule’s surface, on the contrary. Nevertheless, it may still be available for protelytic enzymes.
A possible explication for low perforin content
As mentioned, NK cells from CFS patients have a low perforin content (Maher, et al., 2005)↑, associated to a high perforin gene expression (Brenu, et al., 2011)↑. NK perforin content has been evaluated with the use of a phycoerythrin labeled anti-perforin antibody with an assay described in a previous study (Maher, et al., 2002)↑. According to my hypothesis, perforin is digested with a cleavage between residues 329 and 353. If the epitope of the anti-perforin antibody used by Maher and colleagues to detect NK perforin included part of this peptide, the antibody would fail to detect perforin, even if truncated forms of this enzyme were present. Unfortunately, we don’t know the epitope of human perforin bound by the anti-perforin Ab used by Maher and colleagues. We thus predicted both linear and conformational B cells-epitopes by using Ellipro (Ponomarenko, et al., 2008)↑, a free software which calculates the level of protrusion (protrusion index) of each residues from protein central ellipsoids of inertia which include a specified portion of the protein itself, according to Thornton’s algorithm (Thornton, et al., 1986)↑. As the structure of human perforin has not been experimentally determined yet, we allowed Ellipro to predict a 3D model using as a template the experimental structure of murine perforin (↑), found in 2010 with X-ray method and a resolution of 2,75 Å (Law, et al., 2010)↑. Through this analysis, we found several linear and conformation epitopes, with our peptide present in three of them. In particular, residues 329-335 are included in a big conformational epitope. This means that this peptide is both an available target for proteolytic enzymes and a possible epitope for an anti-perforin antibody. In Figure 2 you can appreciate the surface exposure of peptide 329-335 in a model built by ModBase using murine perforin (↑) as a template. This model has been downloaded in .PDB format, then converted to ASN1 data, using VAST search (↑) page and hence read with Cn3D 4.3.1.
We have proposed the hypothesis that the low level of intracellular perforin in NK cells from ME/CFS patients is due to the same proteolytic activity which underlays cleavage of Rnase-L in this patients group. We have proposed a possible proteases target shared by Rnase-L and perforin and we have shown how this theory matches with available experimental data. As for the reasons of this increased proteolytic activity, we quote Demettre and colleagues, who noted that: “proteases play an important role in numerous physiological responses and, in particular, in inflammation and apoptosis” (Demettre, et al., 2002)↑.
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