bioRxiv (Cold Spring Harbor Laboratory), Apr 16, 2024
Silk fibers, with their highly ordered structure and mechanically superb properties, are produced... more Silk fibers, with their highly ordered structure and mechanically superb properties, are produced in arthropod glands at minimal energy input and ambient conditions, a remarkable feat yet to be achieved synthetically. Due to the high instability and shear sensitivity of the silk protein feedstock, understanding silk fiber formation has been largely limited to in-vitro studies of certain gland sections, offering only a fragmented view of this process. Here, we monitor the whole silk feedstock processing in-situ, at the nano-to micron-scales, through imaging its progressive macromolecular assemblies and phase transitions along the entire Bombyx mori silkworm silk gland. This is done by combining state-of-the-art microscopy techniques, such as cryogenic sample preparation, fixation, and imaging. Our work reveals that fibroin assembles into micron-sized spherical storage "compartments" in the posterior and middle gland sections, a state that ensures its stability and avoids premature fibrillation. These compartments undergo several structural transformations along the gland and eventually disassemble at the entry to the anterior section, before the silk feedstock spinning begins. The spinning itself commences via a series of structural transitions, from the alignment of protein chains in liquid feedstock, through the formation of several fibrillated nano-structures and, in the final stage, a network of cross-linked nano-bundles, which determines the structure and properties of the final microfiber. Importantly, the length of the anterior section of the silk gland enables such gradual and balanced structural transitions. This direct imaging of silk's natural formation process can help formulate a template for the transformation of fibrillar proteins into synthetic bio-fibers. Dedication This work is dedicated to the memory of Dr. Eyal Shimoni, who was a valued colleague and a dear friend. Eyal was a vital part of this research and was essential in shaping its direction. He will be deeply missed for his intellect, mindfulness, creativity, and unwavering dedication to scientific development. Though he is no longer with us, his influence and spirit continue to inspire us in our scientific pursuits. May his passion for discovery and commitment to excellence live on through this work. .
Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of function... more Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the asymmetrical cross-sectional area is a perfect circle. The thermal treatments impact not only the mechanics of the degummed fibroin fibers but also the structural configuration of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To overcome these limitations, we have developed a non-thermal surface treatment that removes the sericin gum layer while preserving the molecular structure of the fibroin protein, and consequently, maintaining the hierarchical integrity of the exposed fibroin microfiber. The precise determination of the initial cross-sectional area of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the newly developed surface treatment raised the Young's modulus and tensile strength by increasing the extent of the fibers' crystallinity by approximately 40% and 50%, respectively, without compromising their strain. Furthermore, we have shown that this treatment facilitated the production of highpurity soluble silk protein with rheological and self-assembly characteristics comparable to those of native silk feedstock, initially stored in the animal's silk gland. The developed approaches benefits both the development of silk-based materials with tailored properties and the proper mechanical characterization of asymmetrical fibrous biological materials made of natural building blocks.
Silk fibers’ unique mechanical properties have made them a desirable material for various applica... more Silk fibers’ unique mechanical properties have made them a desirable material for various applications, from medical to optical materials and even in sensing. Yet, to date, no synthetic method has come close to reproducing this remarkably strong biomaterial due to the complexity and insufficient understanding of the mechanism of silk fiber formation. While ions are known to play a key role in the production of natural silk fiber, how they do so has thus far eluded discovery. Here we report that a broad composition of metal ions guides structural transformations in the silk fibroin protein inside the silkworm silk gland. By using a combination of cryo-electron microscopy techniques coupled with elemental analysis, we followed the changes in the composition and spatial localization of metal ions inside the silk gland. We observed that ions are homogenously dispersed during the initial stages of silk secretion and storage inside the silk gland, but once the fibers are spun, the ions de...
Protein folding is crucial for biological activity. Proteins’ failure to fold correctly underlies... more Protein folding is crucial for biological activity. Proteins’ failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into β-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein’s structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme’s structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the β-sheet fold of fibrillar lysozyme resulting in their structural transformation into...
Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, ... more Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, no synthetic method has come close to reproducing the properties of natural silk, due to the complexity and insufficient understanding of the mechanism of the silk fiber formation. Here, we use a combination of bulk analytical techniques and nanoscale analytical methods, including nano-infrared spectroscopy coupled with atomic force microscopy, to probe the structural characteristics directly, transitions, and evolution of the associated mechanical properties of silk protein species corresponding to the supramolecular phase states inside the silkworm’s silk gland. We found that the key step in silk-fiber production is the formation of nanoscale compartments that guide the structural transition of proteins from their native fold into crystalline β-sheets. Remarkably, this process is reversible. Such reversibility enables the remodeling of the final mechanical characteristics of silk mater...
Cyclic 6-membered aromatic compounds such as benzene and azabenzenes (pyridine, pyridazine, and p... more Cyclic 6-membered aromatic compounds such as benzene and azabenzenes (pyridine, pyridazine, and pyrazine) are known to be light-sensitive, affording, in particular, the Dewar benzene type of intermediates. Pyridine is known to provide the only Dewar pyridine intermediate that undergoes reversible ring-opening. We found that irradiation of photosensitive gels prepared from poly(4-vinyl pyridine) and pyridine at 254 or 312 nm leads to pyridine ring-opening and subsequent formation of 5-amino-2,4-pentadienals. We show that this light-induced process is only partially reversible, and that the photogenerated aminoaldehyde and aminoaldehyde-pending groups undergo self-condensation to produce cross-linked, conjugated oligomers that absorb light in the visible spectrum up to the near-infrared range. Such a sequence of chemical reactions results in the formation of gel with two distinct morphologies: spheres and fiber-like matrices. To gain deeper insight into this process, we prepared poly(...
Peptides and proteins have evolved to self‐assemble into supramolecular entities through a set of... more Peptides and proteins have evolved to self‐assemble into supramolecular entities through a set of non‐covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli‐responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo‐SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addi...
The process of amyloid nanofibril formation has broad implications including the generation of th... more The process of amyloid nanofibril formation has broad implications including the generation of the strongest natural materials, namely silk fibers, and their major contribution to the progression of many degenerative diseases. The key question that remains unanswered is whether the amyloidogenic nature, which includes the characteristic H-bonded β-sheet structure and physical characteristics of protein assemblies, can be modified via controlled intervention of the molecular interactions. Here we show that tailored changes in molecular interactions, specifically in the H-bonded network, do not affect the nature of amyloidogenic fibrillation, and even have minimal effect on the initial nucleation events of self-assembly. However, they do trigger changes in networks at a higher hierarchical level, namely enhanced 2D packaging which is rationalized by the 3D hierarchy of β-sheet assembly, leading to variations in fibril morphology, structural composition and, remarkably, nanomechanical ...
Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader ssilencing (SL... more Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader ssilencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRPSC7.1/7.2. Co-silencing of SKCRPSC7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRPSC7.1 is involved in SLS signaling a...
The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animal... more The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modi...
Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. ... more Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both specie...
In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-deli... more In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the "ultrasonic emulsification method". It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA-RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA-BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA-BSA complex are reported.
The spread of antibiotic‐resistant bacteria and parasites calls for the development of new therap... more The spread of antibiotic‐resistant bacteria and parasites calls for the development of new therapeutic strategies with could potentially reverse this trend. Here, a proposal is presented to exploit a sonochemical method to restore the antibiotic activity of tetracycline (TTCL) against resistant bacteria by converting the antibiotic into a nanoparticulate form. The demonstrated sonochemical method allows nanoscale TTCL assembly to be driven by supramolecular hydrogen bond formation, with no further modification to the antibiotic's chemical structure. It is shown that tetracycline nanoparticles (TTCL NPs) can act as antibacterial agents, both against TTCL sensitive and against resistant bacterial strains. Moreover, the synthesized antibiotic nanoparticles (NPs) can act as effective gene‐silencing agents through the use of a TTCL repressor in Trypanosome brucei parasites. It is demonstrated that the NPs are nontoxic to human cells and T. brucei parasites and are able to release the...
SummaryApproximately 20% of plant genes possess upstream open‐reading frames (uORFs). The effect ... more SummaryApproximately 20% of plant genes possess upstream open‐reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense‐mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF‐peptide, which excluded peptide‐specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. ...
In trypanosomes, mRNas are processed by trans-splicing; in this process, a common exon, the splic... more In trypanosomes, mRNas are processed by trans-splicing; in this process, a common exon, the spliced leader, is added to all mRNas from a small RNa donor, the spliced leader RNa (sL RNa). however, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TsR1 and TsR1IP, implicated in trans-splicing in Trypanosoma brucei. Depletion of these factors by RNai suggested their role in both cisand trans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNas was changed, suggesting that these proteins have a role in regulating only a subset of T. brucei mRNas. Massspectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNa stability, translation, and rRNa processing. We further demonstrate changes in the stability of mRNa as a result of depletion of the two TsR proteins. In addition, rRNa defects were observed under the depletion of U2aF35, TsR1, and TsR1IP, but not sF1, suggesting involvement of sR proteins in rRNa processing.
Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make th... more Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. DNA nanotechnology is a subfield of nanotechnology that seeks to use the unique molecular-recognition properties of DNA and other nucleic acids to create novel, controllable structures of DNA. Chemically, DNA consists of two long polymers. DNA is normally a linear molecule, in that its axis is unbranched. Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. The influence of ultrasonic waves on native DNA molecules has been previously reported. In those studies, it was shown that 2 min of ultrasonication of an aqueous solution of DNA splits the DNA helix into fragments; this makes ultrasonication a useful and convenient tool for obtaining DNA fragments on a preparative scale. Here, we show, for the first time, that ultrasonic waves can be used to convert native DNA molecules to extremely stable DNA nanoparticles (DNA nanospheres, DNs). In addition, the genetic information that was encoded in the DNA nanospheres was successfully delivered to competent cells and to human U2OS cancer cells, and expressed in competent (E. coli) cells. Our fundamental research on the synthesis and characterization of sonochemically produced DNA nanospheres provides an estimate of the efficiency of the sonochemical process in converting the native DNA molecules to biologically active DNA nanospheres. Ultrasonic emulsification is a well-known process that occurs in biphasic systems. Emulsification is necessary for microcapsule formation. Micrometer-sized gasor liquid-filled micro/ nanospheres can be produced from various kinds of proteins such as bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), and from combination of proteins. The mechanism of the sonochemical formation of protein microspheres (PMs) has been discussed previously. The microspheres are formed by chemically crosslinking cysteine residues, which undergo oxidation by HO2 radicals formed around a micron-sized gas bubble or a nonaqueous droplet. The formation of S!S bonds is a direct result of the chemical effects of the ultrasound radiation on an aqueous medium. Our reaction involved the sonication of an aqueous solution of DNA and dodecane (or soya oil) in a 50 mL sonication cell for three minutes. Five kinds of DNA were used in this work: 1) genomic DNA extracted from cells, 2) genomic DNA extracted from leaves, 3) DNA plasmid, 4) linear DNA extracted from DNA plasmid, and 5) single-stranded DNA. For all five DNAs and for both organic solvents we obtained DNA nanospheres. No difference was found between DNA nanospheres filled with dodecane and those filled with soya oil. We further demonstrate that the denaturing conditions as well as denaturing agents, which are commonly used in DNA isolation, cannot destroy the dsDNA (double-stranded DNA) nanospheres of the four DNA’s, while the nanospheres obtained from ssDNA could disintegrate to re-form the individual starting molecules. The efficiency of the sonochemical method in converting native DNA to DNA nanospheres was analyzed by spectrophotometry (NanoDrop 1000 spectrophotometer). It was found that 73.6% of DNA was converted into nanospheres under air and 96% under argon. It is worth underlining that unlike PMs, which are formed only under air and not under argon, DNA nanospheres are formed under both. In order to be sure that the nanobubbles in the solution after sonication are DNA nanospheres and not a combination of fragmented DNA, EtBr dye solution (commonly used for DNA and RNA detection) was added to the product solution. Nanospheres were colored red; this means that the walls of the nanobubbles consisted of DNA molecules (see the Supporting Information). The morphology of the nanospheres in the solution was determined by using scanning environmental-electron microscopy (E-SEM) and light microscopy (Apo-Tome Zeiss1 microscope). For all five types of DNA, the same spherical structure was observed. In Figure 1A, an E-SEM image of the DNA nanospheres that were produced from DNA type I is presented. The spherical morphology of the nanospheres made of DNA is very similar to that of proteinaceous microspheres, but the diameter of the DNA nanospheres (280 nm) was much smaller than the 2500 nm seen for PMs. A large number of dsDNA (DNA type I) are presented in Figure 1B. The size distribution of three kinds of DN (from DNA types I, III, and V) was examined on a DLS apparatus. When a solution of native DNA molecules was sonicated, the DLS results yielded spheres with an average size varying from 290 to 486 nm. A table summarizing average sizes and electrical charges of DNA nanospheres versus DNA type is presented in the Supporting Information. The DNA nanospheres were found to have an electrical charge of !40.7 mV. The electrical charge (z-potential) of [a] Dr. U.…
bioRxiv (Cold Spring Harbor Laboratory), Apr 16, 2024
Silk fibers, with their highly ordered structure and mechanically superb properties, are produced... more Silk fibers, with their highly ordered structure and mechanically superb properties, are produced in arthropod glands at minimal energy input and ambient conditions, a remarkable feat yet to be achieved synthetically. Due to the high instability and shear sensitivity of the silk protein feedstock, understanding silk fiber formation has been largely limited to in-vitro studies of certain gland sections, offering only a fragmented view of this process. Here, we monitor the whole silk feedstock processing in-situ, at the nano-to micron-scales, through imaging its progressive macromolecular assemblies and phase transitions along the entire Bombyx mori silkworm silk gland. This is done by combining state-of-the-art microscopy techniques, such as cryogenic sample preparation, fixation, and imaging. Our work reveals that fibroin assembles into micron-sized spherical storage "compartments" in the posterior and middle gland sections, a state that ensures its stability and avoids premature fibrillation. These compartments undergo several structural transformations along the gland and eventually disassemble at the entry to the anterior section, before the silk feedstock spinning begins. The spinning itself commences via a series of structural transitions, from the alignment of protein chains in liquid feedstock, through the formation of several fibrillated nano-structures and, in the final stage, a network of cross-linked nano-bundles, which determines the structure and properties of the final microfiber. Importantly, the length of the anterior section of the silk gland enables such gradual and balanced structural transitions. This direct imaging of silk's natural formation process can help formulate a template for the transformation of fibrillar proteins into synthetic bio-fibers. Dedication This work is dedicated to the memory of Dr. Eyal Shimoni, who was a valued colleague and a dear friend. Eyal was a vital part of this research and was essential in shaping its direction. He will be deeply missed for his intellect, mindfulness, creativity, and unwavering dedication to scientific development. Though he is no longer with us, his influence and spirit continue to inspire us in our scientific pursuits. May his passion for discovery and commitment to excellence live on through this work. .
Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of function... more Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the asymmetrical cross-sectional area is a perfect circle. The thermal treatments impact not only the mechanics of the degummed fibroin fibers but also the structural configuration of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To overcome these limitations, we have developed a non-thermal surface treatment that removes the sericin gum layer while preserving the molecular structure of the fibroin protein, and consequently, maintaining the hierarchical integrity of the exposed fibroin microfiber. The precise determination of the initial cross-sectional area of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the newly developed surface treatment raised the Young's modulus and tensile strength by increasing the extent of the fibers' crystallinity by approximately 40% and 50%, respectively, without compromising their strain. Furthermore, we have shown that this treatment facilitated the production of highpurity soluble silk protein with rheological and self-assembly characteristics comparable to those of native silk feedstock, initially stored in the animal's silk gland. The developed approaches benefits both the development of silk-based materials with tailored properties and the proper mechanical characterization of asymmetrical fibrous biological materials made of natural building blocks.
Silk fibers’ unique mechanical properties have made them a desirable material for various applica... more Silk fibers’ unique mechanical properties have made them a desirable material for various applications, from medical to optical materials and even in sensing. Yet, to date, no synthetic method has come close to reproducing this remarkably strong biomaterial due to the complexity and insufficient understanding of the mechanism of silk fiber formation. While ions are known to play a key role in the production of natural silk fiber, how they do so has thus far eluded discovery. Here we report that a broad composition of metal ions guides structural transformations in the silk fibroin protein inside the silkworm silk gland. By using a combination of cryo-electron microscopy techniques coupled with elemental analysis, we followed the changes in the composition and spatial localization of metal ions inside the silk gland. We observed that ions are homogenously dispersed during the initial stages of silk secretion and storage inside the silk gland, but once the fibers are spun, the ions de...
Protein folding is crucial for biological activity. Proteins’ failure to fold correctly underlies... more Protein folding is crucial for biological activity. Proteins’ failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into β-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein’s structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme’s structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the β-sheet fold of fibrillar lysozyme resulting in their structural transformation into...
Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, ... more Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, no synthetic method has come close to reproducing the properties of natural silk, due to the complexity and insufficient understanding of the mechanism of the silk fiber formation. Here, we use a combination of bulk analytical techniques and nanoscale analytical methods, including nano-infrared spectroscopy coupled with atomic force microscopy, to probe the structural characteristics directly, transitions, and evolution of the associated mechanical properties of silk protein species corresponding to the supramolecular phase states inside the silkworm’s silk gland. We found that the key step in silk-fiber production is the formation of nanoscale compartments that guide the structural transition of proteins from their native fold into crystalline β-sheets. Remarkably, this process is reversible. Such reversibility enables the remodeling of the final mechanical characteristics of silk mater...
Cyclic 6-membered aromatic compounds such as benzene and azabenzenes (pyridine, pyridazine, and p... more Cyclic 6-membered aromatic compounds such as benzene and azabenzenes (pyridine, pyridazine, and pyrazine) are known to be light-sensitive, affording, in particular, the Dewar benzene type of intermediates. Pyridine is known to provide the only Dewar pyridine intermediate that undergoes reversible ring-opening. We found that irradiation of photosensitive gels prepared from poly(4-vinyl pyridine) and pyridine at 254 or 312 nm leads to pyridine ring-opening and subsequent formation of 5-amino-2,4-pentadienals. We show that this light-induced process is only partially reversible, and that the photogenerated aminoaldehyde and aminoaldehyde-pending groups undergo self-condensation to produce cross-linked, conjugated oligomers that absorb light in the visible spectrum up to the near-infrared range. Such a sequence of chemical reactions results in the formation of gel with two distinct morphologies: spheres and fiber-like matrices. To gain deeper insight into this process, we prepared poly(...
Peptides and proteins have evolved to self‐assemble into supramolecular entities through a set of... more Peptides and proteins have evolved to self‐assemble into supramolecular entities through a set of non‐covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli‐responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo‐SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addi...
The process of amyloid nanofibril formation has broad implications including the generation of th... more The process of amyloid nanofibril formation has broad implications including the generation of the strongest natural materials, namely silk fibers, and their major contribution to the progression of many degenerative diseases. The key question that remains unanswered is whether the amyloidogenic nature, which includes the characteristic H-bonded β-sheet structure and physical characteristics of protein assemblies, can be modified via controlled intervention of the molecular interactions. Here we show that tailored changes in molecular interactions, specifically in the H-bonded network, do not affect the nature of amyloidogenic fibrillation, and even have minimal effect on the initial nucleation events of self-assembly. However, they do trigger changes in networks at a higher hierarchical level, namely enhanced 2D packaging which is rationalized by the 3D hierarchy of β-sheet assembly, leading to variations in fibril morphology, structural composition and, remarkably, nanomechanical ...
Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader ssilencing (SL... more Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader ssilencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRPSC7.1/7.2. Co-silencing of SKCRPSC7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRPSC7.1 is involved in SLS signaling a...
The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animal... more The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modi...
Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. ... more Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both specie...
In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-deli... more In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the "ultrasonic emulsification method". It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA-RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA-BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA-BSA complex are reported.
The spread of antibiotic‐resistant bacteria and parasites calls for the development of new therap... more The spread of antibiotic‐resistant bacteria and parasites calls for the development of new therapeutic strategies with could potentially reverse this trend. Here, a proposal is presented to exploit a sonochemical method to restore the antibiotic activity of tetracycline (TTCL) against resistant bacteria by converting the antibiotic into a nanoparticulate form. The demonstrated sonochemical method allows nanoscale TTCL assembly to be driven by supramolecular hydrogen bond formation, with no further modification to the antibiotic's chemical structure. It is shown that tetracycline nanoparticles (TTCL NPs) can act as antibacterial agents, both against TTCL sensitive and against resistant bacterial strains. Moreover, the synthesized antibiotic nanoparticles (NPs) can act as effective gene‐silencing agents through the use of a TTCL repressor in Trypanosome brucei parasites. It is demonstrated that the NPs are nontoxic to human cells and T. brucei parasites and are able to release the...
SummaryApproximately 20% of plant genes possess upstream open‐reading frames (uORFs). The effect ... more SummaryApproximately 20% of plant genes possess upstream open‐reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense‐mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF‐peptide, which excluded peptide‐specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. ...
In trypanosomes, mRNas are processed by trans-splicing; in this process, a common exon, the splic... more In trypanosomes, mRNas are processed by trans-splicing; in this process, a common exon, the spliced leader, is added to all mRNas from a small RNa donor, the spliced leader RNa (sL RNa). however, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TsR1 and TsR1IP, implicated in trans-splicing in Trypanosoma brucei. Depletion of these factors by RNai suggested their role in both cisand trans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNas was changed, suggesting that these proteins have a role in regulating only a subset of T. brucei mRNas. Massspectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNa stability, translation, and rRNa processing. We further demonstrate changes in the stability of mRNa as a result of depletion of the two TsR proteins. In addition, rRNa defects were observed under the depletion of U2aF35, TsR1, and TsR1IP, but not sF1, suggesting involvement of sR proteins in rRNa processing.
Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make th... more Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. DNA nanotechnology is a subfield of nanotechnology that seeks to use the unique molecular-recognition properties of DNA and other nucleic acids to create novel, controllable structures of DNA. Chemically, DNA consists of two long polymers. DNA is normally a linear molecule, in that its axis is unbranched. Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. The influence of ultrasonic waves on native DNA molecules has been previously reported. In those studies, it was shown that 2 min of ultrasonication of an aqueous solution of DNA splits the DNA helix into fragments; this makes ultrasonication a useful and convenient tool for obtaining DNA fragments on a preparative scale. Here, we show, for the first time, that ultrasonic waves can be used to convert native DNA molecules to extremely stable DNA nanoparticles (DNA nanospheres, DNs). In addition, the genetic information that was encoded in the DNA nanospheres was successfully delivered to competent cells and to human U2OS cancer cells, and expressed in competent (E. coli) cells. Our fundamental research on the synthesis and characterization of sonochemically produced DNA nanospheres provides an estimate of the efficiency of the sonochemical process in converting the native DNA molecules to biologically active DNA nanospheres. Ultrasonic emulsification is a well-known process that occurs in biphasic systems. Emulsification is necessary for microcapsule formation. Micrometer-sized gasor liquid-filled micro/ nanospheres can be produced from various kinds of proteins such as bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), and from combination of proteins. The mechanism of the sonochemical formation of protein microspheres (PMs) has been discussed previously. The microspheres are formed by chemically crosslinking cysteine residues, which undergo oxidation by HO2 radicals formed around a micron-sized gas bubble or a nonaqueous droplet. The formation of S!S bonds is a direct result of the chemical effects of the ultrasound radiation on an aqueous medium. Our reaction involved the sonication of an aqueous solution of DNA and dodecane (or soya oil) in a 50 mL sonication cell for three minutes. Five kinds of DNA were used in this work: 1) genomic DNA extracted from cells, 2) genomic DNA extracted from leaves, 3) DNA plasmid, 4) linear DNA extracted from DNA plasmid, and 5) single-stranded DNA. For all five DNAs and for both organic solvents we obtained DNA nanospheres. No difference was found between DNA nanospheres filled with dodecane and those filled with soya oil. We further demonstrate that the denaturing conditions as well as denaturing agents, which are commonly used in DNA isolation, cannot destroy the dsDNA (double-stranded DNA) nanospheres of the four DNA’s, while the nanospheres obtained from ssDNA could disintegrate to re-form the individual starting molecules. The efficiency of the sonochemical method in converting native DNA to DNA nanospheres was analyzed by spectrophotometry (NanoDrop 1000 spectrophotometer). It was found that 73.6% of DNA was converted into nanospheres under air and 96% under argon. It is worth underlining that unlike PMs, which are formed only under air and not under argon, DNA nanospheres are formed under both. In order to be sure that the nanobubbles in the solution after sonication are DNA nanospheres and not a combination of fragmented DNA, EtBr dye solution (commonly used for DNA and RNA detection) was added to the product solution. Nanospheres were colored red; this means that the walls of the nanobubbles consisted of DNA molecules (see the Supporting Information). The morphology of the nanospheres in the solution was determined by using scanning environmental-electron microscopy (E-SEM) and light microscopy (Apo-Tome Zeiss1 microscope). For all five types of DNA, the same spherical structure was observed. In Figure 1A, an E-SEM image of the DNA nanospheres that were produced from DNA type I is presented. The spherical morphology of the nanospheres made of DNA is very similar to that of proteinaceous microspheres, but the diameter of the DNA nanospheres (280 nm) was much smaller than the 2500 nm seen for PMs. A large number of dsDNA (DNA type I) are presented in Figure 1B. The size distribution of three kinds of DN (from DNA types I, III, and V) was examined on a DLS apparatus. When a solution of native DNA molecules was sonicated, the DLS results yielded spheres with an average size varying from 290 to 486 nm. A table summarizing average sizes and electrical charges of DNA nanospheres versus DNA type is presented in the Supporting Information. The DNA nanospheres were found to have an electrical charge of !40.7 mV. The electrical charge (z-potential) of [a] Dr. U.…
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Papers by Dror Eliaz