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Drug Delivery Dec 2020Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can...
Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20-1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.
Topics: Cells, Cultured; Drug Compounding; HEK293 Cells; Humans; Leukocytes, Mononuclear; Particle Size; Protamines; RNA, Messenger; Transfection
PubMed: 32804028
DOI: 10.1080/10717544.2020.1790692 -
Nucleic Acids Research Jun 2020Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA...
Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.
Topics: DNA; Microscopy, Atomic Force; Nucleic Acid Conformation; Pliability; Protamines
PubMed: 32392345
DOI: 10.1093/nar/gkaa365 -
The Journal of Extra-corporeal... Mar 2020Without anticoagulation, cardiopulmonary bypass would not have developed over the last nearly 60 years into one of the most influential innovations in medicine; without... (Review)
Review
Without anticoagulation, cardiopulmonary bypass would not have developed over the last nearly 60 years into one of the most influential innovations in medicine; without the ability to reverse anticoagulation, cardiac surgery might not have become the common intervention, which is now practiced globally. Despite the recent breathtaking developments in extracorporeal technology, heparin and protamine remain the pillars of anticoagulation and its reversal until this day. However, there is still much controversy in particular about protamine dosing regimens. A number of recent publications investigating various approaches to dosing protamine have rekindled this debate. This review is seeking to capture the current thinking about protamine dosing after cessation of cardiopulmonary bypass.
Topics: Animals; Anticoagulants; Cardiopulmonary Bypass; Heparin; Heparin Antagonists; Humans; Protamines
PubMed: 32280146
DOI: 10.1182/ject-1900038 -
Biomolecules May 2022Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing for 24 h to 1, 10, and 100 nM...
Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of , , and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both and / encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of
Topics: Animals; Chromium; Male; Mytilus; Protamines; Spermatogenesis
PubMed: 35625627
DOI: 10.3390/biom12050700 -
Reproduction in Domestic Animals =... Mar 2020Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively...
Protamines substitute DNA-binding histones during late spermatogenesis in sperm nucleus. Stallion sperm contains all three variants of these arginine-rich and positively charged nuclear proteins (P1, P2 and P3). Two variants of protamine-2, that is P2 and P3, constitute approximately 15% of the entire protamine content. Also, the ratio of protamine-1 to protamine-2 varies among different mammalian species, and abnormal protamine ratios and protamine content are correlated with male infertility. In this study, changes in protamine mRNA abundance for all three protamines were investigated in stallion sperm during cryopreservation. Twelve ejaculates were collected from six sexually mature stallions. Sperm samples were divided into two parts for total mRNA extraction: one as fresh and the other as cryopreserved sample. Levels of three protamine transcripts were determined by real-time reverse transcriptase polymerase chain reaction. Results of relative expression showed that cryopreservation can significantly alter protamine transcripts: protamine 2 was downregulated, while protamine 3 was upregulated in cryopreserved samples relative to the control. Changes in protamine 1 were not significant after cryopreservation. This study is the first to evaluate changes in mRNA abundance of protamine genes in stallion sperm following cryopreservation. Such evaluations are important in finding transcriptomic markers for success in fertilization and assisted reproductive techniques.
Topics: Animals; Cryopreservation; Horses; Male; Protamines; RNA, Messenger; Real-Time Polymerase Chain Reaction; Semen Preservation; Spermatozoa
PubMed: 31885108
DOI: 10.1111/rda.13615 -
International Journal of Molecular... Jun 2020Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal...
Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal activity. Antimicrobial peptides and proteins (AMPs), alternative antibacterial agents, are expected to overcome the bacterial resistance. The aim of this study was to develop a new intelligent material in bone tissue engineering based on protamine-loaded hydroxyapatite (protamine/HAp) that uses AMPs rather than antibiotics. It was found that the adsorption of protamine to HAp followed the Langmuir adsorption model and was due to electrostatic and/or hydrophobic interactions. In vitro bacterial adhesion and growth on protamine/HAp was inhibited in a protamine dose-dependent manner. Adherent bacteria exhibited an aberrant morphology for high dosages of protamine/HAp, resulting in the formation of large aggregates and disintegration of the membrane. The released protamine from protamine/HAp also prevented the growth of planktonic bacteria in vitro. However, a high dosage of protamine from powders at loading concentrations over 1000 μg·mL induced a cytotoxic effect in vitro, although those exhibited no apparent cytotoxicity in vivo. These data revealed that protamine/HAp (less than 1000 μg·mL) had both antimicrobial activity and biocompatibility and can be applied for bone substitutes in orthopedic fields.
Topics: Adsorption; Anti-Infective Agents; Bacteria; Bacterial Adhesion; Biofilms; Bone Substitutes; Bone and Bones; Cell Line; Durapatite; Humans; Materials Testing; Microbial Viability; Osteoblasts; Plankton; Protamines; Tissue Engineering
PubMed: 32575446
DOI: 10.3390/ijms21124368 -
Reproduction (Cambridge, England) May 2016In mammals, male germ cells differentiate from haploid round spermatids to flagella-containing motile sperm in a process called spermiogenesis. This process is distinct... (Review)
Review
In mammals, male germ cells differentiate from haploid round spermatids to flagella-containing motile sperm in a process called spermiogenesis. This process is distinct from somatic cell differentiation in that the majority of the core histones are replaced sequentially, first by transition proteins and then by protamines, facilitating chromatin hyper-compaction. This histone-to-protamine transition process represents an excellent model for the investigation of how epigenetic regulators interact with each other to remodel chromatin architecture. Although early work in the field highlighted the critical roles of testis-specific transcription factors in controlling the haploid-specific developmental program, recent studies underscore the essential functions of epigenetic players involved in the dramatic genome remodeling that takes place during wholesale histone replacement. In this review, we discuss recent advances in our understanding of how epigenetic players, such as histone variants and histone writers/readers/erasers, rewire the haploid spermatid genome to facilitate histone substitution by protamines in mammals.
Topics: Animals; Epigenesis, Genetic; Histones; Humans; Male; Protamines; Spermatogenesis
PubMed: 26850883
DOI: 10.1530/REP-15-0562 -
European Journal of Pharmaceutics and... Oct 2023Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly...
Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly due to their poor stability, limited cellular uptake, and poor target specificity. Cell-penetrating peptides (CPPs), a group of positively charged peptides, represent a breakthrough as delivery systems for macromolecules. In the present study, we used two types of nanoparticles which differ in the type of CPP used for their manufacturing. The first type is composed of protamine, an arginine rich CPP, which is highly positively charged. The arginine residues are able to form electrostatic interactions with miRNAs, stabilize them, and deliver them to cells. The second type is composed of the N-Ter peptide (also known as MPG), an amphipathic peptide rich in lysine. The positively charged parts of the N-Ter peptide electrostatically stabilize miRNAs, whereas its amphipathic character allows it to successfully traverse cell membranes. We used miRNA-27a, a negative regulator of adipogenesis, to form nanoparticles with the peptides and traced their uptake in 3T3-L1 preadipocytes. Motivated by the lengthy discourse regarding the uptake mechanism of CPPs, the focus of our study was to analyse and understand the internalization of proticles (protamine nanoparticles) and N-Ter complexes. The nanoparticles were characterized regarding size, size distribution, and zeta potential, and their cytotoxicity was tested in 3T3-L1 cells. The uptake studies were performed by varying the experimental conditions such as time, concentration, and temperature, as well as by applying different inhibitors of endocytosis. Furthermore, we assessed the biological effect of miRNA-27a on the pro-adipogenic machinery. The obtained data have shown that protamine and the N-Ter peptide form positively charged nanoparticles through non-covalent complexation. The uptake of proticles and N-Ter complexes was found to be dependent on time, concentration, and temperature, and different uptake pathways were discovered to be involved in the internalization of the different nanoparticles. Furthermore, both types of nanoparticles induced the anti-adipogenic effect of miRNA-27a, demonstrating that this approach can be used as a novel miRNA replacement therapy in the treatment of obesity and obesity-related disorders.
Topics: MicroRNAs; Cell-Penetrating Peptides; Drug Delivery Systems; Endocytosis; Protamines; Arginine
PubMed: 37666365
DOI: 10.1016/j.ejpb.2023.08.019 -
WIREs Mechanisms of Disease Mar 2023Male germ cells undergo an extreme but fascinating process of chromatin remodeling that begins in the testis during the last phase of spermatogenesis and continues... (Review)
Review
Male germ cells undergo an extreme but fascinating process of chromatin remodeling that begins in the testis during the last phase of spermatogenesis and continues through epididymal sperm maturation. Most of the histones are replaced by small proteins named protamines, whose high basicity leads to a tight genomic compaction. This process is epigenetically regulated at many levels, not only by posttranslational modifications, but also by readers, writers, and erasers, in a context of a highly coordinated postmeiotic gene expression program. Protamines are key proteins for acquiring this highly specialized chromatin conformation, needed for sperm functionality. Interestingly, and contrary to what could be inferred from its very specific DNA-packaging function across protamine-containing species, human sperm chromatin contains a wide spectrum of protamine proteoforms, including truncated and posttranslationally modified proteoforms. The generation of protamine knock-out models revealed not only chromatin compaction defects, but also collateral sperm alterations contributing to infertile phenotypes, evidencing the importance of sperm chromatin protamination toward the generation of a new individual. The unique features of sperm chromatin have motivated its study, applying from conventional to the most ground-breaking techniques to disentangle its peculiarities and the cellular mechanisms governing its successful conferment, especially relevant from the protein point of view due to the important epigenetic role of sperm nuclear proteins. Gathering and contextualizing the most striking discoveries will provide a global understanding of the importance and complexity of achieving a proper chromatin compaction and exploring its implications on postfertilization events and beyond. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology.
Topics: Male; Humans; Chromatin; Semen; Spermatozoa; Infertility, Male; Protamines
PubMed: 36181449
DOI: 10.1002/wsbm.1588 -
Andrology May 2019Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging...
BACKGROUND
Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa.
OBJECTIVES
The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis.
MATERIALS AND METHODS
For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry.
RESULTS
We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results.
CONCLUSION
Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.
Topics: Animals; Cattle; Cell Nucleus; Epididymis; Gene Expression; Male; Protamines; RNA, Messenger; Spermatozoa; Testis
PubMed: 30920782
DOI: 10.1111/andr.12610