Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell-specific manner.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

The impact of the nelfinavir resistance-conferring mutation D30N on the susceptibility of HIV-1 subtype B to other protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease mutation D30N is exclusively selected by the protease inhibitor (PI) nelfinavir and confers resistance to this drug. We demonstrate that D30N increases the susceptibility to saquinavir (SQV) and amprenavir in HIV-1 subtype B isolates and that the N88D mutation in a D30N background neutralizes this effect. D30N also suppresses indinavir (IDV) resistance caused by the M46I mutation. Interestingly, in patients with viruses originally containing the D30N mutation who were treated with IDV or SQV, the virus either reversed this mutation or acquired N88D, suggesting an antagonistic effect of D30N upon exposure to these PIs. These findings can improve direct salvage drug treatment in resource limited countries where subtype B is epidemiologically important and extend the value of first and second line PIs in these populations.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs

The hepatitis C virus (HCV) NS3 protease has been one of the molecular targets of new therapeutic approaches. Its genomic sequence variability in Brazilian HCV isolates is poorly documented. To obtain more information on the magnitude of its genetic diversity, 114 Brazilian HCV samples were sequenced and analysed together with global reference sequences. Genetic distance (d) analyses revealed that subtype 1b had a higher degree of heterogeneity (d = 0.098) than subtypes 1a (d = 0.060) and 3a (d = 0.062). Brazilian isolates of subtype 1b were distributed in the phylogenetic tree among sequences from other countries, whereas most subtype 1a and 3a sequences clustered into a single branch. Additional characterisation of subtype 1a in clades 1 and 2 revealed that all but two Brazilian subtype 1a sequences formed a distinct and strongly supported (approximate likelihood-ratio test = 93) group of sequences inside clade 1. Moreover, this subcluster inside clade 1 presented an unusual phenotypic characteristic in relation to the presence of resistance mutations for macrocyclic inhibitors. In particular, the mutation Q80K was found in the majority of clade 1 sequences, but not in the Brazilian isolates. These data demonstrate that Brazilian HCV subtypes display a distinct pattern of genetic diversity and reinforce the importance of sequence information in future therapeutic approaches.

Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin

Summary: The mammalian epidermis is a pluristratified epithelium composed of 90% keratinocytes, and its main function is to serve as barrier for the body. The epithelial sodium channel (ENaC), formed by three homologous subunits α, β and γ is found in a variety of epithelia including epidermis. Previous studies showed that ENaC modulates different aspects of epidermal differentiation, such as synthesis of differentiation-specific proteins and lipid secretion. ENaC plays also a critical role in sodium homeostasis of renal and pulmonary epithelia, and its activity is thereby well controlled by hormones and non-hormonal factors, such as the serine protease CAP1 (channel-activating protease 1), also termed prostasin encoded by Prss8 gene. Serine proteases are proteolytic enzymes involved in numerous physiological and pathological processes in the epidermis. In order to evaluate the role of β and γENaC in epidermis, we analyzed the skin phenotype of β and γENaC null mutant (βENaC-/- and γENaC-/-) mice in comparison with the phenotype of αENaC-deficient mice. Furthermore, keratin14-specific CAP1-deficient mice (Prss8lox/Δ /K14-Cre) were generated in order to unveil the role of the serine protease CAP1 in epidermal development and function. This study reveals that the skin phenotype of βENaC and γENaC null mutant mice is less severe than the one of αENaC-deficient mice. However, all these mice present a common premature lipid secretion in the mid-granular layer of the epidermis. Further, the composition of the lipids of the stratum corneum in αENaC-deficient mice is strongly altered, suggesting that epidermal barrier function is compromised. K14-specific CAP1-deficient newborn mice are born at the expected Mendelian ratio, but die soon after birth, showing that CAP1 is required for postnatal survival. The epidermis of these mice exhibits striking malformations of the stratum corneum showing hyperkeratosis. These defects seriously affect both inward and outward epidermal barrier function, leading to rapid and fatal dehydration. As in αENaC-deficient mice, the lipid composition of the stratum corneum of K14-specific CAP1-deficient mice is disturbed. Furthermore, lack of CAP1 leads to the selective loss of filaggrin monomers, important for keratins aggregation and skin moisturization, and to an increased of aberrant profilaggrin precursors. In conclusion, both ENaC and CAP1 expression in the epidermis are crucial for keratinocyte differentiation processes and/or barrier function. Since the abnormalities in K14-specific CAP1-deficient mice resemble key features of human skin ichthyosis, in particular Harlequin ichthyosis, the study of ENaC and CAP1 mutant mice might allow new insights into mechanisms underlying skin diseases. Résumé: L'épiderme des mammifères est un épithélium pluristratifié, protégeant le corps contre les perturbations extérieures et la déshydratation. Le canal épithélial à sodium (ENaC), formé de trois sous-unités α, β et γ, est exprimé dans de nombreux épithélia, comme l'épiderme. Des études ont montré que l'absence de la sous-unité αENaC modulait différents aspects de la différenciation des kératinocytes de l'épiderme, comme la synthèse de protéines spécifiques ou la sécrétion de lipides dans la couche granulaire de l'épiderme. ENaC joue également un rôle crucial dans l'homéostasie du sodium dans les épithélia électriquement étanches, comme l'épithélium rénal ou pulmonaire. L'activité de ENaC est par conséquent finement régulée, en partie par des hormones, mais aussi par des facteurs non-hormonaux, telle que la sérine protéase CAP1 (« channel-activating protease 1 >>) (nommée également prostasine et codée par le gène Prss8). Le but de ce travail a donc été d'étudier le rôle des sous-unités β et γENaC dans l'épiderme en comparaison avec celui de la sous-unité α en utilisant des souris mutantes βENaC-/- et γENaC-/-. Dans un deuxième temps, le phénotype d'une souris chez qui CAP1 a été spécifiquement invalidé dans l'épiderme (Prsslox/Δ/K14-Cre) a été analysé, dans le but de mettre en évidence le rôle de cette protéase dans l'épiderme. Comme déjà montré pour les souris αENaC-/-, la sécrétion des lipides dans la couche granulaire de l'épiderme des souris βENaC-/- et γENaC-/- est prématurée. Cependant, l'hyperplasie et l'expression anormale des protéines marqueurs de la différenciation présents chez les souris αENaC-/- n'ont pas été observés dans l'épiderme des souris βENaC-/- et γENaC-/-. La composition lipidique de la couche cornée des souris αENaC-/- est fortement altérée suggérant que la fonction de barrière de l'épiderme de ces souris est compromise. Les souris mutantes CAP1 ont quant à elles révélé des malformations sévères de leur couche cornée, affectant la fonction de barrière de leur épiderme et conduisant à la mort de ces souris par déshydratation quelques jours après leur naissance. De plus, la composition en lipides de la couche cornée ainsi que la taille des cellules cornées, les cornéocytes, de ces souris sont modifiées par rapport aux souris contrôles. L'invalidation de la protéine CAP1 dans l'épiderme conduit aussi à la perte de la filaggrine, une protéine cruciale pour l'agrégation des kératines dans la couche cornée et le maintien du niveau d'hydratation de la peau, et à l'accumulation de ses précurseurs. En conclusion, l'expression de ENaC et de CAP1 est cruciale pour la différenciation de l'épiderme et/ou sa fonction de barrière. De plus, le phénotype des souris mutantes CAP1 présente des caractéristiques qui ressemblent à celles observées dans certaines pathologies humaines cutanées, comme l'ichthyose d'Harlequin. L'étude des souris mutantes ENaC et CAP1 pourrait donc apporter de nouvelles connaissances dans les mécanismes impliqués dans l'ichthyose d'Harlequin ou d'autres maladies de la peau chez l'homme. Résumé tout public: La peau est le plus grand organe vital du corps humain. Sa fonction principale est de protéger le corps comme une barrière, contre les agressions extérieures et la déshydratation. De nombreuses maladies de la peau résultent d'une perte de fonction de cette barrière. Bien que les pathologies cutanées soient très bien décrites, leur cause génétique n'est en général pas encore connue. La souris est alors un modèle de choix pour la recherche fondamentale. En effet, grâce aux progrès récents de la science, le génome de la souris peut aujourd'hui être modifié dans le but d'étudier le rôle de nombreuses protéines. Dans différents organes, comme le rein et le poumon, le canal épithélial à sodium (ENaC), composé de trois sous-unités protéiques homologues (α, β, et γ), joue un rôle essentiel dans la réabsorption du sodium. L'activité de ENaC est régulée par de nombreux facteurs hormonaux et non-hormonaux, telle que la protéase CAP1 (« channel-activating protease 1 »). L'invalidation de la sous-unité αENaC chez la souris a permis de montrer que dans la peau, le canal ENaC est impliqué dans la différenciation des cellules de l'épiderme et la croissance des poils. Durant ce travail, le phénotype des souris chez qui la protéine βENaC, γENaC ou CAP1 a été invalidée (souris mutantes), a été étudié dans le but de mieux comprendre le rôle des sous-unités du canal ENaC et de son régulateur CAP1 dans la peau. Les résultats de ce projet ont montré que les souris mutantes βENaC et γENaC présentent un épiderme anormal avec une synthèse prématurée de lipides dans la couche granulaire, suggérant l'implication de ENaC dans la fonction de barrière de la peau. De plus, quand CAP1 est invalidé de manière totale chez les souris, le développement embryonnaire est perturbé et ces souris meurent avant la naissance. CAP1 a donc été invalidé spécifiquement dans l'épiderme des souris. Ces souris mutantes « épiderme-spécifique » naissent normalement, mais meurent peu après la naissance de déshydratation. La couche superficielle de l'épiderme, appelée couche cornée, de ces souris est malformée et ne confère plus à la peau sa fonction de barrière. De plus, les composants de la couche cornée, les cellules cornées entourées de lipides, sont sévèrement altérés. Le phénotype de ces souris ressemble aux caractéristiques présentes chez les patients atteints d'ichthyoses, en particulier l'ichthyose d'Harlequin. En conclusion, le canal ENaC ainsi que son régulateur CAP1 jouent un rôle clé dans les processus de différenciation de l'épiderme et/ou de sa fonction de barrière. De plus, les souris mutantes pour CAP1 et ENaC se révéleront peut-être comme des modèles appropriés dans l'étude de l'ichthyose d'Harlequin ou d'autres maladies cutanées.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Determinants for membrane association of the hepatitis C virus NS2 protease domain.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is required for HCV polyprotein processing and particle assembly. It comprises an N-terminal membrane domain and a C-terminal, cytosolically oriented protease domain. Here, we demonstrate that the NS2 protease domain itself associates with cellular membranes. A single charged residue in the second α-helix of the NS2 protease domain is required for proper membrane association, NS2 protein stability, and efficient HCV polyprotein processing.

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease.

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.

Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.

Through their capacity to sense danger signals and to generate active interleukin-1β (IL-1β), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1β, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1β was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.

Study of chemical modification of alkaline lignin by the glyoxalation reaction

In this study, glyoxalated alkaline lignins with a non-volatile and non-toxic aldehyde, which can be obtained from several natural resources, namely glyoxal, were prepared and characterized for its use in wood adhesives. The preparation method consisted of the reaction of lignin with glyoxal under an alkaline medium. The influence of reaction conditions such as the molar ratio of sodium hydroxide-to-lignin and reaction time were studied relative to the properties of the prepared adducts. The analytical techniques used were FTIR and 1H-NMR spectroscopies, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results from both the FTIR and 1H-NMR spectroscopies showed that the amount of introduced aliphatic hydroxyl groups onto the lignin molecule increased with increasing reaction time and reached a maximum value at 10 h, and after they began to decrease. The molecular weights remained unchanged until 10 h of reaction time, and then started to increase, possibly due to the repolymerization reactions. DSC analysis showed that the glass transition temperature (Tg) decreased with the introduction of glyoxal onto the lignin molecule due to the increase in free volume of the lignin molecules. TGA analysis showed that the thermal stability of glyoxalated lignin is not influenced and remained suitable for wood adhesives. Compared to the original lignin, the improved lignin is reactive and a suitable raw material for adhesive formula

The suitability of steam exploded vitis vinifera and alkaline lignin for the manufacture of fiberboard

The main objective of this study was to explore the suitability of Vitis vinifera as a raw material and alkaline lignin as a natural binder for fiberboard manufacturing. In the first step, Vitis vinifera was steam- exploded through a thermo-mechanical vapor process in a batch reactor, and the obtained pulp was dried, ground, and pressed to produce the boards. The effects of pretreatment factors and pressing conditions on the chemical composition of the fibers and the physico-mechanical properties of binderless fiberboards were evaluated, and the conditions that optimize these properties were found. A response surface method based on a central composite design and multiple-response optimization was used. The variables studied and their respective variation ranges were: pretreatment temperature (Tr: 190-210ºC), pretreatment time (tr: 5-10 min), pressing temperature (Tp: 190-210ºC), pressing pressure (Pp: 8-16MPa), and pressing time (tp: 3-7min). The results of the optimization step show that binderless fiberboards have good water resistance and weaker mechanical properties. In the second step, fiberboards based on alkaline lignin and Vitis vinifera pulp produced at the optimal conditions determined for binderless fiberboards were prepared and their physico-mechanical properties were tested. Our results show that the addition of about 15% alkaline lignin leads to the production of fiberboards that fully meet the requirements of the relevant standard specifications

Characterization of alkaline lignins for use in penol-formaldehyde and epoxy resins

Besides polyurethanes and polyesters, phenolic and epoxy resins are the most prominent applications for technical lignins in thermosetting materials. To evaluate the potential application of lignin raw materials in phenol formaldehyde and epoxy resins, three types of alkaline lignins were characterized in terms of their structures and thermal properties. The lignin samples analyzed were kraft lignin (LIG-1), soda–rice straw lignin (LIG-2), and soda-wheat straw lignin (LIG-3). FTIR and 1H-NMR methods were used to determine their structure. Gel permeation chromatography (GPC) was used to determine the molecular weight distribution (MWD). Differential scanning calorimetry (DSC) was used to measure the glass transition temperature (Tg), and thermogravimetric analysis (TGA) to determine the thermal stability of lignin samples. Results showed that kraft lignin (LIG-1) has moderate hydroxyl-group content, is rich in G-type units, and has good thermal stability. These properties make it more suitable for direct use in phenol formaldehyde resins, and it is therefore a good raw material for this purpose. The alkaline soda-rice straw lignin (LIG-2) with a high hydroxyl-group content and excellent thermal stability is most suited to preparing lignin-based epoxy resin

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Extended bottom-up proteomics with secreted aspartic protease Sap9.

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7kDa on average) peptides in bottom-up proteomics to about twice longer (~3.2kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, ~1h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. BIOLOGICAL SIGNIFICANCE: Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (<3kDa) peptides, which may exceed the analytical capabilities of liquid chromatography and mass spectrometry. Information on localization of protein modifications may also be affected by the small size of typically produced peptides. On the other hand, advances in high-resolution mass spectrometry and liquid chromatography have created an intriguing opportunity of improving proteome analysis by gradually increasing the size of enzymatically-derived peptides in MS-based bottom-up proteomics. Bioinformatics has already confirmed the envisioned advantages of such approach. The remaining bottle-neck is an enzyme that could produce longer peptides. Here, we report on the characterization of a possible candidate enzyme, Sap9, which may be considered for producing longer, e.g., 3-7kDa, peptides and lead to a development of extended bottom-up proteomics.