Mechanoresponsive Protein Crystals for NADH Recycling in Multicycle Enzyme Reactions.

NAD(H)-dependent enzymes play a crucial role in the biosynthesis of pharmaceuticals and fine chemicals, but the limited recyclability of the NAD(H) cofactor hinders its more general application. Here, we report the generation of mechano-responsive PEI-modified Cry3Aa protein crystals and their use for NADH recycling over multiple reaction cycles. For demonstration of its practical utility, a complementary Cry3Aa protein particle containing genetically encoded and co-immobilized formate dehydrogenase for NADH regeneration and leucine dehydrogenase for catalyzing the NADH-dependent l-tert-leucine (l-tert-Leu) biosynthesis has been produced. When combined with the PEI-modified Cry3Aa crystal, the resultant reaction system could be used for the efficient biosynthesis of l-tert-Leu for up to 21 days with a 10.5-fold improvement in the NADH turnover number.

Pyrrolysine-Inspired in Cellulo Synthesis of an Unnatural Amino Acid for Facile Macrocyclization of Proteins.

Macrocyclization has been touted as an effective strategy to enhance the in vivo stability and efficacy of protein therapeutics. Herein, we describe a scalable and robust system based on the endogenous biosynthesis of a noncanonical amino acid coupled to the pyrrolysine translational machinery for the generation of lasso-grafted proteins. The in cellulo biosynthesis of the noncanonical amino acid d-Cys-ε-Lys was achieved by hijacking the pyrrolysine biosynthesis pathway, and then, its genetical incorporation into proteins was performed using an optimized PylRS/tRNAPyl pair and cell line. This system was then applied to the structurally inspired cyclization of a 23-mer therapeutic P16 peptide engrafted on a fusion protein, resulting in near-complete cyclization of the target cyclic subunit in under 3 h. The resulting cyclic P16 peptide fusion protein possessed much higher CDK4 binding affinity than its linear counterpart. Furthermore, a bifunctional bicyclic protein harboring a cyclic cancer cell targeting RGD motif on the one end and the cyclic P16 peptide on the other is produced and shown to be a potent cell cycle arrestor with improved serum stability.

Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis.

We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent in vitro activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, FM09h, was highly active with IC50 of 0.3 µM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of FM09h remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of FM09h (10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).

In Vivo Enzyme Entrapment in a Protein Crystal.

Cry3Aa is a protein that forms crystals naturally in the bacterium Bacillus thuringiensis. Here we report that coexpression of Cry3Aa and a Proteus mirabilis lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst. These characteristics, along with its greatly simplified method of isolation, highlight the potential of Cry3Aa crystal-mediated enzyme entrapment for use in biocatalysis and other biotechnological applications.

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

Pyrrolysine-inspired protein cyclization.

The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of "site-specific" non-canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole-like topology. As a proof of concept, we fused the well-studied RGD peptide to the C terminus of an mCherry reporter protein. Previous studies have shown that cyclization of the RGD peptide enhances its internalization into cells compared to its linear counterpart. The cellular uptake efficiencies of mCherry-cyclo(RGD), mCherry-linear(RGD), and wild-type mCherry determined by flow cytometry follow the trends expected, thereby confirming the feasibility and potential utility of this cyclization approach.

A Targeted and Protease-Activated Genetically Encoded Melittin-Containing Particle for the Treatment of Cutaneous and Visceral Leishmaniasis.

Intracellular infections are difficult to treat, as pathogens can take advantage of intracellular hiding, evade the immune system, and persist and multiply in host cells. One such intracellular parasite, Leishmania, is the causative agent of leishmaniasis, a neglected tropical disease (NTD), which disproportionately affects the world's most economically disadvantaged. Existing treatments have relied mostly on chemotherapeutic compounds that are becoming increasingly ineffective due to drug resistance, while the development of new therapeutics has been challenging due to the variety of clinical manifestations caused by different Leishmania species. The antimicrobial peptide melittin has been shown to be effective in vitro against a broad spectrum of Leishmania, including species that cause the most common form, cutaneous leishmaniasis, and the most deadly, visceral leishmaniasis. However, melittin's high hemolytic and cytotoxic activity toward host cells has limited its potential for clinical translation. Herein, we report a design strategy for producing a melittin-containing antileishmanial agent that not only enhances melittin's leishmanicidal potency but also abrogates its hemolytic and cytotoxic activity. This therapeutic construct can be directly produced in bacteria, significantly reducing its production cost critical for a NTD therapeutic. The designed melittin-containing fusion crystal incorporates a bioresponsive cathepsin linker that enables it to specifically release melittin in the phagolysosome of infected macrophages. Significantly, this targeted approach has been demonstrated to be efficacious in treating macrophages infected with L. amazonensis and L. donovani in cell-based models and in the corresponding cutaneous and visceral mouse models.

A click-and-release pyrrolysine analogue.

What's the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites.

A Bioresponsive Genetically Encoded Antimicrobial Crystal for the Oral Treatment of Helicobacter Pylori Infection.

Helicobacter pylori (H. pylori) causes infection in the stomach and is a major factor for gastric carcinogenesis. The application of antimicrobial peptides (AMPs) as an alternative treatment to traditional antibiotics is limited by their facile degradation in the stomach, their poor penetration of the gastric mucosa, and the cost of peptide production. Here, the design and characterization of a genetically encoded H. pylori-responsive microbicidal protein crystal Cry3Aa-MIIA-AMP-P17 is described. This designed crystal exhibits preferential binding to H. pylori, and when activated, promotes the targeted release of the AMP at the H. pylori infection site. Significantly, when the activated Cry3Aa-MIIA-AMP-P17 crystals are orally delivered to infected mice, the Cry3Aa crystal framework protects its cargo AMP against degradation, resulting in enhanced in vivo efficacy against H. pylori infection. Notably, in contrast to antibiotics, treatment with the activated crystals results in minimal perturbation of the mouse gut microbiota. These results demonstrate that engineered Cry3Aa crystals can serve as an effective platform for the oral delivery of therapeutic peptides to treat gastrointestinal diseases.

Insights from the Structure of an Active Form of Bacillus thuringiensis Cry5B.

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112-698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27-698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112-698) structure, and also exhibits a change in the conformation of residues 112-140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112-140 in its alternate α-helix conformation. Finally, based on the Cry5B(27-698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity.

Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of TP53 gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape. Given the high mutation frequency of TP53 in TNBC cells, restoration of p53 function could be a potential strategy to overcome their resistance to anti-programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) therapy. Herein, we have assessed the use of Pos3Aa crystal-based platform to mediate the intracellular delivery of p53 protein to restore p53 activity in p53 null tumors and consequently augment anti-PD-1 activity. METHODS: The efficiency of Pos3Aa-p53 crystals in delivering p53 protein was evaluated using confocal imaging, immunofluorescence staining, flow cytometry and RNA-seq. The ability of Pos3Aa-p53 crystals to remodel tumor microenvironment was investigated by examining the markers of immunogenic cell death (ICD) and the expression of PD-L1, indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase 2 and type I interferon (IFN). Finally, both unilateral and bilateral 4T1 tumor mouse models were utilized to assess the efficacy of Pos3Aa-p53 crystal-mediated p53 restoration in enhancing the antitumor activity of ICIs. T cells in tumor tissues and spleens were analyzed, and the in vivo biosafety of the Pos3Aa-p53 crystal/anti-PD-1 antibody combination was also evaluated. RESULTS: Delivery of p53 protein into p53-null TNBC 4T1 cells via Pos3Aa-p53 crystals restored the p53 activity, and therefore led to the induction of ICD, activation of type I IFN signaling and upregulation of PD-L1 expression. Pos3Aa-p53 crystals significantly enhanced T cell infiltration and activation in 4T1 tumors, thereby sensitizing them to anti-PD-1 therapy. The combination of Pos3Aa-p53 crystals with anti-PD-1 antibody also induced a systemic antitumor immunity resulting in the inhibition of distal tumor growth with minimal toxicity. CONCLUSION: This study validates that p53 restoration can be an effective approach to overcome ICI resistance and demonstrates that intracellular delivery of p53 protein can be an efficient, safe and potentially universal strategy to restore p53 activity in tumors carrying TP53 mutation.

SIB-BLAST: a web server for improved delineation of true and false positives in PSI-BLAST searches.

A SIB-BLAST web server (http://sib-blast.osc.edu) has been established for investigators to use the SimpleIsBeautiful (SIB) algorithm for sequence-based homology detection. SIB was developed to overcome the model corruption frequently observed in the later iterations of PSI-BLAST searches. The algorithm compares resultant hits from the second iteration to the final iteration of a PSI-BLAST search, calculates the figure of merit for each 'overlapped' hit and re-ranks the hits according to their figure of merit. By validating hits generated from the last profile against hits from the first profile when the model is least corrupted, the true and false positives are better delineated, which in turn, improves the accuracy of iterative PSI-BLAST searches. Notably, this improvement to PSI-BLAST comes at minimal computational cost as SIB-BLAST utilizes existing results already produced in a PSI-BLAST search.

Efficient intracellular delivery of p53 protein by engineered protein crystals restores tumor suppressing function in vivo.

Direct delivery of proteins into cells holds significant potential for basic research and drug development. However, the poor endosomal escape of conventional delivery strategies remains a challenge, thus limiting the clinical translation of many protein therapeutics. Herein, we report that engineered Cry3Aa protein (Pos3Aa) crystals formed naturally within Bacillus thuringiensis can serve as a vehicle for efficient cytosolic delivery of bioactive proteins. We showed that Pos3Aa-mediated delivery of tumor suppressor p53 protein, a promising therapeutic candidate found to be inactivated in nearly half of human cancers, resulted in the restoration of p53 function in p53-deficient cancer cells, and thereby sensitized them to 5-fluorouracil chemotherapy as demonstrated in in vitro and in vivo models. Our results validate that Pos3Aa crystals can be a robust and effective platform for the cytosolic delivery of effector proteins, and suggest that efficient uptake and endosomal escape could be critical for efficacious p53 protein-based cancer therapy.

A SUMO1-Derived Peptide Targeting SUMO-Interacting Motif Inhibits α-Synuclein Aggregation.

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-ß component domain of α-synuclein has been implicated in modulating its aggregation. Herein, we report the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55)), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models.

Efficient encapsulation of functional proteins into erythrocytes by controlled shear-mediated membrane deformation.

Red blood cells (RBCs) are attractive carriers of biomolecular payloads due to their biocompatibility and the ability to shelter their encapsulated cargo. Commonly employed strategies to encapsulate payloads into RBCs, such as hypotonic shock, membrane fusion or electroporation, often suffer from low throughput and unrecoverable membrane impairment. This work describes an investigation of a method to encapsulate protein payloads into RBCs by controlling membrane deformation either transiently or extendedly in a microfluidic channel. Under the optimized conditions, the loading efficiency of enhanced green fluorescent protein into mouse RBCs increased was about 2.5- and 4-fold compared to that with osmotic entrapment using transient and extended deformation, respectively. Significantly, mouse RBCs loaded with human arginase exhibit higher enzymatic activity and membrane integrity compared to their counterparts loaded by osmotic entrapment. These features together with the fact that this shear-mediated encapsulation strategy allows loading with physiological buffers highlight the key advantages of this approach compared to traditional osmotic entrapment.

Cytosolic delivery of CDK4/6 inhibitor p16 protein using engineered protein crystals for cancer therapy.

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins into cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

The effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

Insight into the substrate length restriction of M32 carboxypeptidases: characterization of two distinct subfamilies.

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily.

Simple is beautiful: a straightforward approach to improve the delineation of true and false positives in PSI-BLAST searches.

MOTIVATION: The deluge of biological information from different genomic initiatives and the rapid advancement in biotechnologies have made bioinformatics tools an integral part of modern biology. Among the widely used sequence alignment tools, BLAST and PSI-BLAST are arguably the most popular. PSI-BLAST, which uses an iterative profile position specific score matrix (PSSM)-based search strategy, is more sensitive than BLAST in detecting weak homologies, thus making it suitable for remote homolog detection. Many refinements have been made to improve PSI-BLAST, and its computational efficiency and high specificity have been much touted. Nevertheless, corruption of its profile via the incorporation of false positive sequences remains a major challenge. RESULTS: We have developed a simple and elegant approach to resolve the problem of model corruption in PSI-BLAST searches. We hypothesized that combining results from the first (least-corrupted) profile with results from later (most sensitive) iterations of PSI-BLAST provides a better discriminator for true and false hits. Accordingly, we have derived a formula that utilizes the E-values from these two PSI-BLAST iterations to obtain a figure of merit for rank-ordering the hits. Our verification results based on a 'gold-standard' test set indicate that this figure of merit does indeed delineate true positives from false positives better than PSI-BLAST E-values. Perhaps what is most notable about this strategy is that it is simple and straightforward to implement.

A pyrrolysine analogue for protein click chemistry.

Ignoring the STOP sign: A pyrrolysine analogue bearing a terminal alkyne was site-specifically incorporated into recombinant calmodulin (CaM) through a UAG codon. The resulting protein was labeled with an azide-containing dye using a copper(I)-catalyzed click reaction. Subsequent application of an orthogonal cysteine tagging method yielded a CaM labeled with two distinct fluorophores that enabled its study by FRET spectroscopy.