Radiotherapy remains one of the main pillars of cancer treatment and is used in more than half of all oncological patients. Despite continuous technological improvements, ionizing radiation inevitably causes damage to surrounding healthy tissues, leading to acute and chronic complications affecting multiple organs, including the skin, mucosa, heart, lungs, bones and gastrointestinal tract. Radiation-induced injuries significantly impair patients’ quality of life, limit therapeutic doses, and represent a major unmet clinical challenge. Hydrogels have emerged as promising biomaterials for managing radiation-induced damage due to their high content of water, tunable mechanics, and ability to mimic the extracellular matrix. Recent innovations have introduced functional systems, including stimuli-responsive, injectable, and bioactive hydrogels, capable of delivering antioxidants, growth factors, or living cells. Unlike traditional material-based reviews, this work proposes a novel classification framework based on the hydrogel’s mechanism of action within the pathophysiology of radiation injury. We evaluate how specific designs, such as ROS-scavenging matrices, barrier-forming injectable shields, and bioactive delivery vehicles, address distinct phases of inflammation and fibrosis. By providing a comprehensive overview of radiation-induced injuries across different organs, this review summarizes current hydrogel-based strategies for both prevention and therapy. We highlight the potential of these mechanistically aligned systems to protect healthy tissues, suppress chronic inflammation, and promote effective tissue regeneration.

Proteasomes catalyze protein degradation in cells and play an integral role in cellular homeostasis. Its activity decreases with age alongside the load of defective proteins, resulting from mutations or oxidative stress-induced damage. Such proteins are prone to aggregation and, if not efficiently degraded, can form toxic oligomers and amyloid plaques. Developing an effective way to activate the proteasome could prevent such pathologies. Designing activators is not easy because they do not bind in the active site, which is well-defined and highly conserved, but away from it. The structures of proteasome complexes with natural activators can help here, but these are large proteins, some even multimeric, whose activity is difficult to replace with a small-molecule compound. Nevertheless, the use of fragments of such proteins makes it possible to accumulate knowledge about the relevance of various structural elements for efficient and selective activation. Here, we presented peptidic activators of the 20S proteasome, which were designed based on both the C-terminal sequence of the yeast proteasome activator, Blm10 protein, and the interactions predicted by molecular modeling. These Blm analogs were able to stimulate human 20S proteasome to more efficiently degrade both small fluorogenic substrates and proteins. The best activators also demonstrated their efficacy in cell lysates. X-ray crystallography indicated that an effective modulator can bind to several sites on the surface of the proteasome without causing permanent structural changes in its immediate vicinity but affecting the active sites.

The development of age-related neurodegenerative diseases is associated with the accumulation of damaged and misfolded proteins. Such proteins are eliminated from cells by proteolytic systems, mainly by 20S proteasomes, whose activity declines with age. Its stimulation has been recognized as a promising approach to delay the onset or ameliorate the symptoms of neurodegenerative disorders. Here we present peptidomimetics that are very effective in stimulating the proteasome in biochemical assays and in cell culture. They are stable in human plasma and capable of penetrating the cell membranes. The activators demonstrated the ability to enhance h20S degradation of α-synuclein and tau, whose aggregates are involved in the development of Parkinson's and Alzheimer's diseases, respectively. The peptidomimetics did not show cytotoxicity to HEK293T and primary hippocampal cells. Additionally, these compounds were highly effective in reducing the amount of phosphorylated α-synuclein aggregates in hippocampal neurons in a mouse embryonic cell model.

Human amylin, called also islet amyloid polypeptide (hIAPP), is the principal constituent of amyloid deposits in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Preventing aggregation, and in particular inhibiting the formation and/or stimulating degradation of toxic amylin oligomers formed early in the process, may reduce the negative effects of T2DM. Such therapeutic intervention may be enabled by activation of the 20S proteasome, a proteolytic system responsible for digesting proteins that are damaged or natively exhibit aggregation tendencies. In this work, we showed that in the lag phase of the aggregation process, soluble oligomers of small size (dimer to heptamer) were present alongside the amylin monomer. These oligomers inhibited the activity of the human 20S proteasome (h20S). To counteract this inhibition, we designed two activators that proved to be effective in restoring the peptidase efficiency of h20S to basal levels and even stimulating the enzyme to degrade the fluorogenic substrate more efficiently. They showed this effect both against isolated h20S and in cell lysate.

IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

Serum amyloid A (SAA) is a small protein consisting of 104 residues and, under physiological conditions, exists mainly in hexameric form. It belongs to the positive acute-phase proteins, which means that its plasma concentration increases rapidly in response to injury, inflammation, and infection. The accumulation of SAA molecules promotes the formation of amyloid aggregates, which deposit extracellularly in many organs, causing their dysfunction. In our previous work, we successfully designed a peptidomimetic that inhibited the aggregation of amyloidogenic SAA fragments. In the present paper, we show how the same inhibitor, named saa3Dip, affects the oligomerization and aggregation processes of MetSAA1.1 protein. The thioflavin T assay showed that saa3Dip inhibited its fibrillization. The measurement of the internal fluorophore fluorescence (Trp) showed differences that occurred in the tertiary structure of MetSAA1.1 in the presence of the inhibitor, which was also confirmed by CD spectra in the aromatic range. FTIR results suggested that saa3Dip could stabilize some fragments of the native structure of MetSAA1.1, which was confirmed by determining the melting temperature (Tm) of the protein–inhibitor complex. AFM images demonstrated that the presence of saa3Dip prevented the formation of large SAA aggregates. Our results suggest that saa3Dip stabilizes the native conformation of MetSAA1.1.

Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens.

The civilization diseases of the 21st century are non-infectious disorders, affecting a large part of modern society. They are associated with the significant development of industry and technology, and hence with environmental pollution and an unhealthy lifestyle. These factors have led to the development of many civilization diseases, which currently include: cardiovascular diseases, respiratory diseases, diabetes, obesity, malignant tumors, gastrointestinal diseases, mental disorders and allergic diseases. The development of technologies, including modern therapies and new drugs, resulted in increase in life expectancy. This creates a global problem of an aging population with an increasing number of diseases of the old age, i.e. dementias. In addition, sedentary lifestyles and changing diets are the reasons why more and more people develop metabolic diseases, as well as neurological and cognitive disorders characterized by progressive damage to nerve cells and dementia. Currently, problem on a global scale is also the growing resistance to existing antimicrobial drugs. Therefore, the scientists face many challenges related to searching for the causes of these diseases, their diagnosis and treatment. Scientific research conducted at the Department of Biomedical Chemistry at the Faculty of Chemistry of the University of Gdańsk is part of this research trend. In this publication, we discuss various research topics with the long-term aim of solving the problems associated with the diseases mentioned above. The following chapters are dedicated to (i) looking for new effective fluorophores with diagnostic and anti-cancer activity; (ii) designing of new compounds with antibacterial and antiviral activity and their synthesis; (iii) investigating the mechanisms of amyloid deposit formation by human cystatin C and possibilities of inhibition of this process; (iv) designing and studies of compounds activating the proteasome with the potential to suppress the development of neurodegenerative diseases; (v) designing peptide fibrils and hydrogels as drug carriers; (vi) searching for peptide inhibitors of immune checkpoint as potential drugs for immunotherapy; (vii) studying the mechanism of action of selected herpesviruses by determining the structure of viral proteins and (viii) studying the composition of natural glycans and glycoconjugates in order to better understand the mechanisms of interaction of bacteria with the environment or with the host

Degradation of misfolded, redundant and oxidatively damaged proteins constitutes one of the cellular processes which are influenced by the 20S proteasome. However, its activity is generally thought to decrease with age which leads to the gradual accumulation of abnormal proteins in cells and their subsequent aggregation. Therefore, increasing proteasomal degradation constitutes a promising strategy to delay the onset of various age-related diseases, including neurodegenerative disorders. In this study we designed and obtained a series of peptidomimetic stimulators of 20S comprising in their sequences the C-terminal fragment of Blm10 activator. Some of the compounds were capable of enhancing the degradation of natively unfolded and oxidatively damaged proteins, such as α-synuclein and enolase, whose applicability as proteasome substrates was evaluated by microscale thermophoresis (MST). Furthermore, they increased the ChT-L activity of the proteasome in HEK293T cell extracts. Our studies indicate that the 20S proteasome-mediated protein substrates hydrolysis may be selectively increased by peptide-based stimulators acting in an allosteric manner. These compounds, after further optimization, may have the potential to counteract proteasome impairment in patients suffering from age-related diseases.

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer’s disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome’s increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a β turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Serum amyloid A variant 1.1 (SAA1.1) is an acute phase protein. In response to injury, inflammation or infection its production increases highly, which may lead to aggregation of the protein and accumulation of its deposits in various organs. Due to the cellular toxicity of the aggregates, as well as the fact that accumulated deposits are a burden that obstructs proper functioning of the affected tissues, it is vital to find a way to suppress the process of pathological aggregates formation. To make this possible, it is necessary to investigate thoroughly the oligomerization process and recognize factors that may influence its course. Some previous studies showed that aromatic interactions are important to the potential of an inhibitor to suppress the aggregation process. In our research we had proved that a five-residue peptide RSFFS (saa1-5) is an efficient inhibitor of aggregation of the most amyloidogenic fragment of SAA1.1, SAA1-12. In the present work the oligomerization and aggregation propensity of SAA1-12 was compared to that of SAA1-27, in order to determine the contribution of the sequence which extends beyond the most amyloidogenic region but encompasses residues reportedly involved in the stabilization of the SAA native conformation. Thioflavin T fluorescence assay, quantitative chromatographic analysis of the insoluble fraction and transmission electron microscopy allowed for a deeper insight into the SAA aggregation process and the morphology of aggregates. Substitutions of Phe3 and/or Phe4 residues in saa1-5 sequence with tryptophan, tyrosine, homophenylalanine, naphthylalanine and β,β-diphenylalanine allowed to study the influence of different aromatic systems on the aggregation of SAA1-12 and SAA1-27, and evaluate these results in relation to hSAA1.1 protein. Our results indicate that compounds with aromatic moieties can affect the course of the aggregation process and change the ratio between the soluble and insoluble aggregates.

In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF. In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays. Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.

One of the approaches in the design of anti-amyloid drugs is to find compounds that are able to hamper self-association of amyloidogenic protein molecules. [...]

The proteasome degrade most of the proteins in eukaryotic cells, thereby controlling the keycellular processes [...]

RNA-recognition motif (RRM) is an RNA-interacting protein domain that plays an important role in the processes of RNA metabolism such as the splicing, editing, export, degradation, and regulation of translation. Here, we present the RNA-recognition motif database (RRMdb), which affords rapid identification and annotation of RRM domains in a given protein sequence. The RRMdb database is compiled from similar to 57 000 collected representative RRM domain sequences, classified into 415 families. Whenever possible, the families are associated with the available literature and structural data. Moreover, the RRM families are organized into a network of sequence similarities that allows for the assessment of the evolutionary relationships between them.