Understanding the Catalytic Mechanism and the Substrate Specificity of an Engineered Gluten Hydrolase by QM/MM Molecular Dynamics and Free Energy Simulations

Celiac sprue, also known as gluten-sensitive enteropathy, is a chronic disease suffered by approximately 1% of the world’s population. Engineered enzymes have been emerging to treat celiac disease by hydrolyzing the pathogenic peptides of gluten. For example, Kuma010 has been studied experimentally and proved to be a promising gluten hydrolase under gastric conditions. However, the detailed catalytic mechanism and the substrate specificity are still unclear. In this paper, quantum-mechanical/molecular-mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to determine the catalytic mechanism, the substrate specificity, and the role of the active-site residues during the reaction. The results given here demonstrate that the Kuma010 has a similar catalytic mechanism but different substrate specificity as wild-type kumamolisin-As. Binding properties of the enzyme (especially mutated residues) and substrate complex are discussed, and activation free energy barriers toward different substrates have also been examined. The computational free energy results are in reasonable agreement with the experimental data. The strategy for developing next-generation gluten hydrolases is discussed

Image_1_Ovarian cancer-associated immune exhaustion involves SPP1+ T cell and NKT cell, symbolizing more malignant progression.pdf

BackgroundOvarian cancer (OC) is highly heterogeneous and has a poor prognosis. A better understanding of OC biology could provide more effective therapeutic paradigms for different OC subtypes.MethodsTo reveal the heterogeneity of T cell-associated subclusters in OC, we performed an in-depth analysis of single-cell transcriptional profiles and clinical information of patients with OC. Then, the above analysis results were verified by qPCR and flow cytometry examine.ResultsAfter screening by threshold, a total of 85,699 cells in 16 ovarian cancer tissue samples were clustered into 25 major cell groups. By performing further clustering of T cell-associated clusters, we annotated a total of 14 T cell subclusters. Then, four distinct single-cell landscapes of exhausted T (Tex) cells were screened, and SPP1 + Tex significantly correlated with NKT cell strength. A large amount of RNA sequencing expression data combining the CIBERSORTx tool were labeled with cell types from our single-cell data. Calculating the relative abundance of cell types revealed that a greater proportion of SPP1 + Tex cells was associated with poor prognosis in a cohort of 371 patients with OC. In addition, we showed that the poor prognosis of patients in the high SPP1 + Tex expression group might be related to the suppression of immune checkpoints. Finally, we verified in vitro that SPP1 expression was significantly higher in ovarian cancer cells than in normal ovarian cells. By flow cytometry, knockdown of SPP1 in ovarian cancer cells could promote tumorigenic apoptosis.ConclusionThis is the first study to provide a more comprehensive understanding of the heterogeneity and clinical significance of Tex cells in OC, which will contribute to the development of more precise and effective therapies.</p

Flow cytometry determination of VapA cell surface localization in <i>Rhodococcus equi</i> using a C-terminal Strep-tagged VapA.

VapA-ST is expressed as a cell surface protein. Flow cytometry of R. equi ΔvapA carrying either pVapA-ST (A, C and D) or pVapA (B). Approximately 7x105 cells were incubated with THE2122; NWSHPQFEK Tag mouse FITC-monoclonal antibody (A, B and D). Cells carrying pVapA were used to assess the level of antibody’s non-specific binding (B). The intrinsic fluorescence of R. equi ΔvapA/pVapA-ST was determined by including a control incubated without the antibody (C). The bacterial sample was digested with Trypsin to remove proteins from the cell surface before incubation with antibody (D). Data are a representative of three independent experiments.</p

This is the S1 Table containing primer sequnecs for RT-PCR shown in Fig 6.

This is the S1 Table containing primer sequnecs for RT-PCR shown in Fig 6.</p

These are the raw images for Figs 3 and 6.

Rhodococcus equi pneumonia is an important cause of mortality in foals worldwide. Virulent equine isolates harbour an 80-85kb virulence plasmid encoding six virulence-associated proteins (Vaps). VapA, the main virulence factor of this intracellular pathogen, is known to be a cell surface protein that creates an intracellular niche for R. equi growth. In contrast, VapC, VapD and VapE are secreted into the intracellular milieu. Although these Vaps share very high degree of sequence identity in the C-terminal domain, the N-terminal domain (N-domain) of VapA is distinct. It has been proposed that this domain plays a role in VapA surface localization but no direct experimental data provides support to such hypothesis. In this work, we employed R. equi 103S harbouring an unmarked deletion of vapA (R. equi ΔvapA) as the genetic background to express C-terminal Strep-tagged Vap-derivatives integrated in the chromosome. The surface localization of these proteins was assessed by flow cytometry using the THE2122;-NWSHPQFEK Tag FITC-antibody. We show that VapA is the only cell surface Vap encoded in the virulence plasmid. We present compelling evidence for the role of the N-terminal domain of VapA on cell surface localization using fusion proteins in which the N-domain of VapD was exchanged with the N-terminus of VapA. Lastly, using an N-terminally Strep-tagged VapA, we found that the N-terminus of VapA is exposed to the extracellular environment. Given the lack of a lipobox in VapA and the exposure of the N-terminal Strep-tag, it is possible that VapA localization on the cell surface is mediated by interactions between the N-domain and components of the cell surface. We discuss the implications of this work on the light of the recent discovery that soluble recombinant VapA added to the extracellular medium functionally complement the loss of VapA.</div

Complementation of <i>R</i>. <i>equi</i> Δ<i>vapA</i> with pVapA-ST and pVapA::VapD-ST.

Murine J774A.1 cells were infected with either R. equi ΔvapA or R. equi ΔvapA carrying either pVapA-ST or pVapA::VapD-ST. Intracellular R. equi were enumerated 0,24 and 48 hours post infection. Intracellular growth is shown as fold changes relative to time zero. Error bars represent the mean and standard deviation. Horizontal lines show the Dunnet’s T3 adjusted P value corrected for multiple comparisons. For simplicity only relevant comparisons are shown.</p

The N-terminus of VapA plays a role in surface localization.

Graphical representation of the fusion proteins containing the N-terminus of VapA (T32-Q84) and the C-terminal domain of VapD that were employed to assess the role of the N-terminus of VapA on surface localization (A). Fusion proteins VapA(SS-N)::VapD-ST (B) and VapD(SS)::VapA(N)::VapD-ST (C). Cell surface proteins were detected with THE2122; NWSHPQFEK Tag mouse FITC-monoclonal antibody (Red) but not detected when cells were digested with trypsin before incubation with the antibody (Black).</p

The N-terminus of VapA is exposed to the extracellular environment.

Detection of the ST-VapA protein on the cell surface by flow cytometry was used to detect accessibility of the N-terminal domain to the extracellular environment (Red). Trypsin digestion of the cells before incubation with the anti-Strep-tag FITC-antibody removed the antibody binding sites (Black).</p

Expression of <i>vap-ST</i> genes in <i>R</i>. <i>equi</i> 103S Δ<i>vapA</i>.

A) Qualitative analysis of Vap-ST transcripts was determined by reverse transcription using the Improm II reverse transcriptase and random 6-mer primers followed by PCR with KAPA2G Fast DNA polymerase as previously described. For the PCR step, a common reverse primer (Vap_ST-rev) targeting the Strep-tag coding sequence was employed together with Vap-specific forward primers (S1 Table) as required for each strain. Lanes: 1) pVapA-ST (172 bp), 2) pVapC-ST (219 bp), 3) pVapD-ST (222 bp), 4) pVapE-ST (134 bp), 5) pVapG-ST (250 bp), 6) pVapH-ST (206 bp) and M) DNA ladder 50 bp (Invitrogen). B) Non-reverse transcriptase control.</p

Addition of a C-terminal Strep-tag does not affect cell surface localisation of VapA.

R. equi ΔvapA/pVapA-ST was grown under vapA inducing conditions, followed by extraction with 2% (v/v) Triton X-114. VapA monoclonal antibodies and Strep-tag HRP conjugate antibodies were used to detect VapA. Lane 1: western blot developed using VapA monoclonal antibodies. Lane 2: western blot developed using Strep-tag HRP conjugate antibodies. Bars on the left indicate the molecular mass in kDa.</p