Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 µM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

Predominant and novel de novo variants in 29 individuals with ALG13 deficiency: Clinical description, biomarker status, biochemical analysis, and treatment suggestions.

Asparagine-linked glycosylation 13 homolog (ALG13) encodes a nonredundant, highly conserved, X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N-linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13-CDG. Twenty-four previously reported ALG13-CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13-CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13-deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13. This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.

Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Matrix metalloproteinase-14 both sheds cell surface neuronal glial antigen 2 (NG2) proteoglycan on macrophages and governs the response to peripheral nerve injury.

Neuronal glial antigen 2 (NG2) is an integral membrane chondroitin sulfate proteoglycan expressed by vascular pericytes, macrophages (NG2-Mφ), and progenitor glia of the nervous system. Herein, we revealed that NG2 shedding and axonal growth, either independently or jointly, depended on the pericellular remodeling events executed by membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14). Using purified NG2 ectodomain constructs, individual MMPs, and primary NG2-Mφ cultures, we demonstrated for the first time that MMP-14 performed as an efficient and unconventional NG2 sheddase and that NG2-Mφ infiltrated into the damaged peripheral nervous system. We then characterized the spatiotemporal relationships among MMP-14, MMP-2, and tissue inhibitor of metalloproteinases-2 in sciatic nerve. Tissue inhibitor of metalloproteinases-2-free MMP-14 was observed in the primary Schwann cell cultures using the inhibitory hydroxamate warhead-based MP-3653 fluorescent reporter. In teased nerve fibers, MMP-14 translocated postinjury toward the nodes of Ranvier and its substrates, laminin and NG2. Inhibition of MMP-14 activity using the selective, function-blocking DX2400 human monoclonal antibody increased the levels of regeneration-associated factors, including laminin, growth-associated protein 43, and cAMP-dependent transcription factor 3, thereby promoting sensory axon regeneration after nerve crush. Concomitantly, DX2400 therapy attenuated mechanical hypersensitivity associated with nerve crush in rats. Together, our findings describe a new model in which MMP-14 proteolysis regulates the extracellular milieu and presents a novel therapeutic target in the damaged peripheral nervous system and neuropathic pain.

The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury.

BACKGROUND: Mechanical pain hypersensitivity associated with physical trauma to peripheral nerve depends on T-helper (Th) cells expressing the algesic cytokine, interleukin (IL)-17A. Fibronectin (FN) isoform alternatively spliced within the IIICS region encoding the 25-residue-long connecting segment 1 (CS1) regulates T cell recruitment to the sites of inflammation. Herein, we analyzed the role of CS1-containing FN (FN-CS1) in IL-17A expression and pain after peripheral nerve damage. METHODS: Mass spectrometry, immunoblotting, and FN-CS1-specific immunofluorescence analyses were employed to examine FN expression after chronic constriction injury (CCI) in rat sciatic nerves. The acute intra-sciatic nerve injection of the synthetic CS1 peptide (a competitive inhibitor of the FN-CS1/α4 integrin binding) was used to elucidate the functional significance of FN-CS1 in mechanical and thermal pain hypersensitivity and IL-17A expression (by quantitative Taqman RT-PCR) after CCI. The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves. RESULTS: Following CCI, FN expression in sciatic nerve increased with the dominant FN-CS1 deposition in endothelial cells, Schwann cells, and macrophages. Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve. CS1 peptide inhibited the LPS- or starvation-stimulated activation of the stress ERK/MAPK pathway in cultured Schwann cells. CONCLUSIONS: After physical trauma to the peripheral nerve, FN-CS1 contributes to mechanical pain hypersensitivity by increasing the number of IL-17A-expressing (presumably, Th17) cells. CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.

Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

Substrate cleavage profiling suggests a distinct function of Bacteroides fragilis metalloproteinases (fragilysin and metalloproteinase II) at the microbiome-inflammation-cancer interface.

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Allosteric Inhibitors of Zika Virus NS2B-NS3 Protease Targeting Protease in "Super-Open" Conformation.

The Zika virus (ZIKV), a member of the Flaviviridae family, is considered a major health threat causing multiple cases of microcephaly in newborns and Guillain-Barré syndrome in adults. In this study, we targeted a transient, deep, and hydrophobic pocket of the "super-open" conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the active site pocket. After virtual docking screening of approximately seven million compounds against the novel allosteric site, we selected the top six candidates and assessed them in enzymatic assays. Six candidates inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations. These six compounds, targeting the selected protease pocket conserved in ZIKV, serve as unique drug candidates and open new opportunities for possible treatment against several flavivirus infections.

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases.

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.