Modeling protease-sensitive human pancreatic lipase mutations in the mouse ortholog.

Pancreatic lipase (PNLIP) is the major lipolytic enzyme secreted by the pancreas. A recent study identified human PNLIP variants P245A, I265R, F300L, S304F, and F314L in European chronic pancreatitis cohorts. Functional analyses indicated that the variants were normally secreted but exhibited reduced stability when exposed to pancreatic proteases. Proteolysis of the PNLIP variants yielded an intact C-terminal domain, while the N-terminal domain was degraded. The protease-sensitive PNLIP phenotype was strongly correlated with chronic pancreatitis, suggesting a novel pathological pathway underlying the disease. To facilitate preclinical mouse modeling, here we investigated how the human mutations affected the secretion and proteolytic stability of mouse PNLIP. We found that variants I265R, F300L, S304F, and F314L were secreted at high levels, while P245A had a secretion defect and accumulated inside the cells. Proteolysis experiments indicated that wild-type mouse PNLIP was resistant to cleavage, while variant I265R was readily degraded by mouse trypsin and chymotrypsin C. Variants F300L, S304F, and F314L were unaffected by trypsin but were slowly proteolyzed by chymotrypsin C. The proteases degraded the N-terminal domain of variant I265R, leaving the C-terminal domain intact. Structural analyses suggested that changes in stabilizing interactions around the I265R mutation site contribute to the increased proteolytic susceptibility of this variant. The results demonstrate that variant I265R is the best candidate for modeling the protease-sensitive PNLIP phenotype in mice.

The human retroviral-like aspartic protease 1 (ASPRV1): From in vitro studies to clinical correlations.

The human retroviral-like aspartic protease 1 (ASPRV1) is a retroviral-like protein that was first identified in the skin due to its expression in the stratum granulosum layer of the epidermis. Accordingly, it is also referred to as skin-specific aspartic protease. Similar to the retroviral polyproteins, the full-length ASPRV1 also undergoes self-proteolysis, the processing of the precursor is necessary for the autoactivation of the protease domain. ASPRV1's functions are well-established at the level of the skin: it is part of the epidermal proteolytic network and has a significant contribution to skin moisturization via the limited proteolysis of filaggrin; it is only natural protein substrate identified so far. Filaggrin and ASPRV1 are also specific for mammalians, these proteins provide unique features for the skins of these species, and the importance of filaggrin processing in hydration is proved by the fact that some ASPRV1 mutations are associated with skin diseases such as ichthyosis. ASPRV1 was also found to be expressed in macrophage-like neutrophil cells, indicating that its functions are not limited to the skin. In addition, differential expression of ASPRV1 was detected in many diseases, with yet unknown significance. The currently known enzymatic characteristics-that had been revealed mainly by in vitro studies-and correlations with pathogenic phenotypes imply potentially important functions in multiple cell types, which makes the protein a promising target of functional studies. In this review we describe the currently available knowledge and future perspective in regard to ASPRV1.

Serine 39 in the GTP-binding domain of Drp1 is involved in shaping mitochondrial morphology.

Continuous fusion and fission are critical for mitochondrial health. In this study, we further characterize the role played by dynamin-related protein 1 (Drp1) in mitochondrial fission. We show that a single amino acid change in Drp1 at position 39 from serine to alanine (S39A) within the GTP-binding (GTPase) domain results in a fused mitochondrial network in human SH-SY5Y neuroblastoma cells. Interestingly, the phosphorylation of Ser-616 and Ser-637 of Drp1 remains unaffected by the S39A mutation, and mitochondrial bioenergetic profile and cell viability in the S39A mutant were comparable to those observed in the control. This leads us to propose that the serine 39 residue of Drp1 plays a crucial role in mitochondrial distribution through its involvement in the GTPase activity. Furthermore, this amino acid mutation leads to structural anomalies in the mitochondrial network. Taken together, our results contribute to a better understanding of the function of the Drp1 protein.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Structural similarity of human papillomavirus E4 and polyomaviral VP4 exhibited by genomic analysis of the common kestrel (Falco tinnunculus) polyomavirus.

Polyomaviruses are widely distributed viruses of birds that may induce developmental deformities and internal organ disorders primarily in nestlings. In this study, polyomavirus sequence was detected in kidney and liver samples of a common kestrel (Falco tinnunculus) that succumbed at a rescue station in Hungary. The amplified 5025 nucleotide (nt) long genome contained the early (large and small T antigen, LTA and STA) and late (viral proteins, VP1, VP2, VP3) open reading frames (ORFs) typical for polyomaviruses. One of the additional putative ORFs (named VP4) showed identical localization with the VP4 and ORF-X of gammapolyomaviruses, but putative splicing sites could not be found in its sequence. Interestingly, the predicted 123 amino acid (aa) long protein sequence showed the highest similarity with human papillomavirus E4 early proteins in respect of the aa distribution and motif arrangement implying similar functions. The LTA of the kestrel polyomavirus shared <59.2% nt and aa pairwise identity with the LTA sequence of other polyomaviruses and formed a separated branch in the phylogenetic tree among gammapolyomaviruses. Accordingly, the kestrel polyomavirus may be the first member of a novel species within the Gammapolyomavirus genus, tentatively named Gammapolyomavirus faltin.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

Molecular Identification and In Silico Protein Analysis of a Novel BCOR-CLGN Gene Fusion in Intrathoracic BCOR-Rearranged Sarcoma.

BCOR (BCL6 corepressor)-rearranged sarcomas (BRSs) are a heterogeneous group of sarcomas previously classified as part of the group of "atypical Ewing" or "Ewing-like" sarcomas, without the prototypical ESWR1 gene translocation. Due to their similar morphology and histopathological features, diagnosis is challenging. The most common genetic aberrations are BCOR-CCNB3 fusion and BCOR internal tandem duplication (ITD). Recently, various new fusion partners of BCOR have been documented, such as MAML3, ZC3H7B, RGAG1, and KMT2D, further increasing the complexity of such tumor entities, although the molecular pathogenetic mechanism remains to be elucidated. Here, we present an index case of intrathoracic BRS that carried a novel BCOR-CLGN (calmegin) gene fusion, exhibited by a 52-year-old female diagnosed initially by immunohistochemistry due to the positivity of a BCOR stain; the fusion was identified by next-generation sequencing and was confirmed by Sanger sequencing. In silico protein analysis was performed to demonstrate the 3D structure of the chimera protein. The physicochemical properties of the fusion protein sequence were calculated using the ProtParam web-server tool. Our finding further broadens the fusion partner gene spectrum of BRS. Due to the heterogeneity, molecular ancillary tests serve as powerful tools to discover these unusual variants, and an in silico analysis of the fusion protein offers an appropriate approach toward understanding the exact pathogenesis of such a rare variant.

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis.

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

BACKGROUND: Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. METHODS: To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. RESULTS: Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein-DNA, protein-protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. CONCLUSIONS: These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.

Early suppression of antiviral host response and protocadherins by SARS-CoV-2 Spike protein in THP-1-derived macrophage-like cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19). The spike protein (S) of SARS-CoV-2 plays a crucial role in mediating viral infectivity; hence, in an extensive effort to curb the pandemic, many urgently approved vaccines rely on the expression of the S protein, aiming to induce a humoral and cellular response to protect against the infection. Given the very limited information about the effects of intracellular expression of the S protein in host cells, we aimed to characterize the early cellular transcriptomic changes induced by expression of the S protein in THP-1-derived macrophage-like cells. Results showed that a wide variety of genes were differentially expressed, products of which are mainly involved in cell adhesion, homeostasis, and most notably, antiviral and immune responses, depicted by significant downregulation of protocadherins and type I alpha interferons (IFNAs). While initially, the levels of IFNAs were higher in the medium of S protein expressing cells, the downregulation observed on the transcriptomic level might have been reflected by no further increase of IFNA cytokines beyond the 5 h time-point, compared to the mock control. Our study highlights the intrinsic pathogenic role of the S protein and sheds some light on the potential drawbacks of its utilization in the context of vaccination strategies.