In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

Peptidylarginine deiminase (PPAD) is a virulence factor unique to pathogenic Porphyromonas species, especially P. gingivalis. Mechanistically, PPAD activity, in conjunction with Arg-specific gingipains, generates protein fragments with citrullinated C-termini. Such polypeptides are potential de novo epitopes that are key drivers of rheumatoid arthritis. This process could underlie the observed clinical association between rheumatoid arthritis and periodontitis. However, the role of PPAD in host colonization by P. gingivalis and, subsequently, in triggering periodontitis is not known. Therefore, the aim of the current study was to delineate the role of PPAD in bacterial biofilm formation, and to define whether adherence to, invasion of, and host responses to bacteria of gingival keratinocytes depend on PPAD activity. We studied these aspects using PPAD-competent and PPAD-incompetent strains of P. gingivalis, and demonstrated that neither biofilm formation nor its composition was affected by PPAD activity. Similarly, flow cytometry revealed that PPAD did not impact the ability of P. gingivalis to adhere to and, subsequently, invade keratinocytes. Network analyses of gene expression patterns, however, revealed a group of host genes that were sensitive to PPAD activity (CXCL8, IL36G, CCL20, and IL1B). These genes can be categorized as potent immune modulators belonging to the interleukin 1 system, or chemoattractants of lymphocytes and neutrophils. Thus, we conclude that PPAD, although it is a potent modulator of the immune response, does not affect bacterial biofilm formation or the ability of P. gingivalis to adhere to and invade gingival epithelial cells.

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.

Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1-TGFβ3) engaged in signaling functions through binding of cognate TGFβ receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFβ2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFβ2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFβ receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGβ2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.

Periodontal disease is one of the most common forms of inflammation. It is currently diagnosed by observing symptoms such as gingival bleeding and attachment loss. However, the detection of biomarkers that precede such symptoms would allow earlier diagnosis and prevention. Aptamers are short oligonucleotides or peptides that fold into three-dimensional conformations conferring the ability to bind molecular targets with high affinity and specificity. Here we report the selection of aptamers that bind specifically to the bacterium Tannerella forsythia, a pathogen frequently associated with periodontal disease. Two aptamers with the highest affinity were examined in more detail, revealing that their binding is probably dependent on mirolysin, a surface-associated protease secreted by the T. forsythia type-9 secretion system. The aptamers showed minimal cross-reactivity to other periodontopathogens and are therefore promising leads for the development of new tools to study the composition of the periodontitis-associated dysbiotic bacteriome as well as inexpensive new diagnostic assays.

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.

Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer, we determined the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method, based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been shown to be implicated in familial early-onset forms of the disease. Using the second relative quantitation method, iTRAQ, we identified 22 differentially regulated proteins, many of which are extracellular proteins known to be involved in proper assembly of the basement membrane in other tissues. In total, 26 differentially regulated proteins were identified, of which 6 proteins were regulated in both methods. These results support that the morphological changes observed in FECD are caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

α2-Macroglobulins (α2Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α2Ms have been studied over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in Drosophila Schneider 2 and human Expi293F cells, which produced pure human α2M (hα2M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα2M was mainly found in the induced form. Shorter hα2M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα2M to recombinant latent human transforming growth factor-β2 (pro-TGF-β2) and bacterial G-related α2M binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic hα2M tetramers. The shorter recombinant hα2M variants interacted after preincubation only. In contrast, pro-TGF-β2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer's disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.

Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct β-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system.

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti-AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786-0-Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786-0-Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786-0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi-target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of Porphyromonas gingivalis and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of P. gingivalis as the keystone pathogen in periodontitis, P. gingivalis QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

MCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored.

Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.