Mature Enzyme Research Articles

Cryo-EM structure of the HIV-1 Pol polyprotein provides insights into virion maturation.

Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo–electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

Expression of beta glucosidase mined from metagenomic DNA data of bacteria in Vietnamese goats’ rumen in \(\textit{Escherichia coli}\) system

Screening and expression of new β-glucosidase genes (bgc) have attracted much attention because of their valuable application in a wide range of industrial areas such as bioethanol production, food and animal feed processing, paper making, and biotechnological processes. Previously, we mined a new bgc gene coding for its mature enzyme (BGC) from metagenomic DNA data of bacteria in Vietnamese goats’ rumen. Based on the NCBI database, the BGC sequence was found to be the highest similarity with β-glucosidase of Bacteroidales bacterium (60.52%). The BGC enzyme was previously annotated to have two domains GH3 and GH31 and highly expressed in E. coli. The aim of this work was to experimentally confirm the predicted gene by expressing and assessing the activity of recombinant BGC from E. coli strains of BL21, Rosetta 1, C43, and SoluBL21. Furthermore, the expression level and solubility of the BGC were also investigated by varying fermentation conditions such as temperature, medium components, and IPTG concentration. The activity of the crude enzyme was identified through substrates including esculin and p-Nitrophenyl-β-D-glucopyranoside (pNPG). The new BGC could be used as potential material for further enzyme characterization.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A).

A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45°C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30°C. The Km and Vmax values of this enzyme were 1.32mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by β-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

Functional expression and characterization of a novel aminopeptidase B from Aspergillus niger in Pichia pastoris.

A novel aminopeptidase B (APB-AN) was identified from Aspergillus niger CGMCC 3.1454 for the first time and was cloned and expressed in Pichia pastoris. The mature enzyme of approximately 100kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40°C, respectively. The enzyme was stable below 40°C and at pH values from 5.0 to 8.0. The K m and V max values were determined to be 0.61mmol/L and 11.45mmol/L/min, respectively, using Arg-pNA as the substrate. APB-AN was inhibited by Cu2+ and Fe2+ and activated by Co2+ and Na+. Most metal chelators (Ca2+, Mg2+ and Mn2+) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid p-nitroanilide (pNA) substrates:Arg-pNA, Lys-pNA, Tyr- pNA, Trp-pNA, Phe-pNA, His-pNA, Ala-pNA, Met-pNA, Leu-pNA, Glu-pNA, Val-pNA, Pro-pNA and Ile-pNA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications.

Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood.

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.

Engineered pro-peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste.

Keratinase is an attractive industrial enzyme that can specifically catalyze keratin waste to obtain value-added products. A challenge to the application of keratinase is improving catalytic capacity to achieve efficient hydrolysis. In this study, we effectively expressed the keratinase gene from Bacillus licheniformis BBE11-1 in Bacillus subtilis WB600 based on pro-peptide engineering. Partial deletion of the pro-peptide sequence and the substitution of amino acid at the pro-peptide cleavage site (P1) suggested that the "chaperone effect"and "cleavage efficiency"of the pro-peptide determine the activity of the mature enzyme. Subsequently, seven target sites that can increase the activity of the mature enzyme by 16%-66% were obtained through the multiple sequence alignment of pro-peptides and site-directed mutation. We further performed combinatorial mutations at six sites based on the design principle of three-codon saturation mutations and obtained mutant 2-D12 (236.8 KU/mg) with a mature enzyme activity of 186% of the original (127.6 KU/mg). Finally, continuous fermentation was carried out in a 5-L bioreactor for 22 h, and the activity of the 2-D12 mature enzyme was increased to 391.6 KU/mg. Most importantly, 2-D12 could degrade more than 90% of feather waste into amino acids and peptides within 12 h with the aid of sulfite.

γ-Glutamyltranspeptidase essential for the metabolism of γ-glutamyl compounds in bacteria and its application.

The enzymatic characteristics of γ-glutamyltranspeptidase were elucidated. The catalytic nucleophile of the enzymatic reaction of Escherichia coli γ-glutamyltranspeptidase was identified as the Oγ of the N-terminal Thr-residue of the small subunit. It was demonstrated that the inactive precursor of γ-glutamyltranspeptidase is processed autocatalytically and intramolecularly into the active heterodimeric mature enzyme via an ester intermediate. The catalytic nucleophile of this processing reaction was identified as the same Oγ atom of the N-terminal Thr-residue of the small subunit. These results were also supported by the three-dimensional structures of the γ-glutamyl enzyme intermediate and of the precursor-mimicked T391A nonprocessable mutant enzyme. Applications of transpeptidation and hydrolysis activities of bacterial γ-glutamyltranspeptidases were developed. Using transpeptidation activity, efficient enzymatic production of useful γ-glutamyl compounds, such as prodrug for Parkinson's disease, theanine and kokumi compound, was enabled. Hydrolysis activity was used as glutaminase and the mutant enzymes gaining glutaryl-7-aminocephalosporanic acid acylase activity were isolated.

The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases.

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.

Origin and Expansion of the Serine Protease Repertoire in the Myelomonocyte Lineage.

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed \u2018clamp-like\u2019 mechanism of binding and inhibition

BackgroundThe zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues.ResultsImmunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site.ConclusionsThe FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a “clamp-like” mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

Schistosoma japonicum cathepsin B2 (SjCB2) facilitates parasite invasion through the skin.

Cercariae invasion of the human skin is the first step in schistosome infection. Proteases play key roles in this process. However, little is known about the related hydrolytic enzymes in Schistosoma japonicum. Here, we investigated the biochemical features, tissue distribution and biological roles of a cathepsin B cysteine protease, SjCB2, in the invasion process of S. japonicum cercariae. Enzyme activity analysis revealed that recombinant SjCB2 is a typical cysteine protease with optimum temperature and pH for activity at 37°C and 4.0, respectively, and can be totally inhibited by the cysteine protease inhibitor E-64. Immunoblotting showed that both the zymogen (50 kDa) and mature enzyme (30.5 kDa) forms of SjCB2 are expressed in the cercariae. It was observed that SjCB2 localized predominantly in the acetabular glands and their ducts of cercariae, suggesting that the protease could be released during the invasion process. The protease degraded collagen, elastin, keratin, fibronectin, immunoglobulin (A, G and M) and complement C3, protein components of the dermis and immune system. In addition, proteomic analysis demonstrated that SjCB2 can degrade the human epidermis. Furthermore, it was showed that anti-rSjCB2 IgG significantly reduced (22.94%) the ability of the cercariae to invade the skin. The cysteine protease, SjCB2, located in the acetabular glands and their ducts of S. japonicum cercariae. We propose that SjCB2 facilitates skin invasion by degrading the major proteins of the epidermis and dermis. However, this cysteine protease may play additional roles in host-parasite interaction by degrading immunoglobins and complement protein.

Cholesterol oxidase from Rhodococcus erythropolis with high specificity toward β-cholestanol and pytosterols.

Two genes (choRI and choRII) encoding cholesterol oxidases belonging to the vanillyl-alcohol oxidase (VAO) family were cloned on the basis of putative cholesterol oxidase gene sequences in the genome sequence data of Rhodococcus erythropolis PR4. The genes corresponding to the mature enzymes were cloned in a pET vector and expressed in Escherichia coli. The two cholesterol oxidases produced from the recombinant E. coli were purified to examine their properties. The amino acid sequence of ChoRI showed significant similarity (57%) to that of ChoRII. ChoRII was more stable than ChoRI in terms of pH and thermal stability. The substrate specificities of these enzymes differed distinctively from one another. Interestingly, the activities of ChoRII toward β-cholestanol, β-sitosterol, and stigmasterol were 2.4-, 2.1-, and 1.7-fold higher, respectively, than those of cholesterol. No cholesterol oxidases with high activity toward these sterols have been reported so far. The cholesterol oxidation products from these two enzymes also differed. ChoRI and ChoRII oxidized cholesterol to form cholest-4-en-3-one and 6β-hydroperoxycholest-4-en-3-one, respectively.

The Efek Enzim Bromelin Buah Nanas (Ananas comosus (L.) Merr) Berbasis Sediaan Gel terhadap Lebar Intertubulus Dentin

Caries tissue cleaning can use Chemo-Mechanical Caries Removal (CMCR) based on proteolytic enzymes that catalyze peptide bonds into simpler compounds. Proteolytic enzymes can be found in mature pineapple bromelain enzymes. The aim of the study is to determine the effect of gel-based bromelain enzyme with concentrations of 8%, 10% and 12% against the width of the intertubulus dentin. The bromelain enzyme is extracted using the Lowry method. Then purified using 80% ethanol and diluting become to concentrations of 8%, 10% and 12%. Diluted bromelain enzymes were formed in gel preparations based on HPMC and applied to the study sample. The results of this study indicated a widening of the intertubulus dentin. This is indicated by the variation of the dentin intertubulus width &gt; 2µm. This widening occurs because the bromelain enzyme can hydrolyze collagen in intertubulus dentin in the absence of alpha-l-antitrypsin. Hydrolysis causes the breaking of hydrogen bonds in the triple helix- shaped tropocollagen to turn into strands of polypeptide chains. The bromelain enzyme with a concentration 10% is more effective than 8% and 12% because it is within the maximum speed limit of the enzyme so that the enzyme is saturated by its substrate and there is a difference in specific enzyme activity in each concentration.

The roles of SDHAF2 and dicarboxylate in covalent flavinylation of SDHA, the human complex II flavoprotein

Mitochondrial complex II, also known as succinate dehydrogenase (SDH), is an integral-membrane heterotetramer (SDHABCD) that links two essential energy-producing processes, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. A significant amount of information is available on the structure and function of mature complex II from a range of organisms. However, there is a gap in our understanding of how the enzyme assembles into a functional complex, and disease-associated complex II insufficiency may result from incorrect function of the mature enzyme or from assembly defects. Here, we investigate the assembly of human complex II by combining a biochemical reconstructionist approach with structural studies. We report an X-ray structure of human SDHA and its dedicated assembly factor SDHAF2. Importantly, we also identify a small molecule dicarboxylate that acts as an essential cofactor in this process and works in synergy with SDHAF2 to properly orient the flavin and capping domains of SDHA. This reorganizes the active site, which is located at the interface of these domains, and adjusts the pKa of SDHAR451 so that covalent attachment of the flavin adenine dinucleotide (FAD) cofactor is supported. We analyze the impact of disease-associated SDHA mutations on assembly and identify four distinct conformational forms of the complex II flavoprotein that we assign to roles in assembly and catalysis.

Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr.

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40°C, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

Oncogenic Gata1 causes stage-specific megakaryocyte differentiation delay.

The megakaryocyte/erythroid transient myeloproliferative disorder (TMD) in newborns with Down syndrome (DS) occurs when Nterminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s embryonic stem cells (ESC) and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ESC-derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of the cell cycle and a reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focused to understand the oncogenic action of GATA1s.

Activation mechanism of plasmepsins, pepsin-like aspartic proteases from Plasmodium, follows a unique trans-activation pathway.

Plasmodium parasites that cause malaria produce plasmepsins (PMs), pepsin-like aspartic proteases that are important antimalarial drug targets due to their role in host hemoglobin degradation. The enzymes are synthesized as inactive zymogens (pro-PMs), and the mechanism of their conversion to the active, mature forms has not been clearly elucidated. Our structural investigations of vacuolar pro-PMs with truncated prosegment (pro-tPMs) reveal that the formation of the S-shaped dimer is their innate property. Further structural studies, biochemical analysis, and molecular dynamics simulations indicate that disruption of the Tyr-Asp loop (121p-4), coordinated with the movement of the loop L1 (237-247) and helix H2 (101p-113p), is responsible for the extension of the pro-mature region (harboring the cleavage site). Consequently, under acidic pH conditions, these structural changes result in the dissociation of the dimers to monomers and the protonation of the residues in the prosegment prompts its unfolding. Subsequently, we demonstrated that the active site of the monomeric pro-tPMs with the unfolded prosegment is accessible for peptide substrate binding; in contrast, the active site is blocked in folded prosegment form of pro-tPMs. Thus, we propose a novel mechanism of auto-activation of vacuolar pro-tPMs that under acidic conditions can form a catalytically competent active site. One monomer cleaves the prosegment of the other one through a trans-activation process, resulting in formation of mature enzyme. As a result, once a mature enzyme is generated, it leads to the complete conversion of all the inactive pro-tPMs to their mature form. DATABASE: Atomic coordinates and structure factors have been submitted in the Protein Data Bank (PDB) under the PDB IDs 6KUB, 6KUC, and 6KUD.