Correct Folding Research Articles

N-glycosylation on Asn-57 is required for the correct HAI-2 protein folding and protease inhibitory activity.

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic β-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in β-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

The Influence of the Selection at the Amino Acid Level on Synonymous Codon Usage from the Viewpoint of Alternative Genetic Codes

Synonymous codon usage can be influenced by mutations and/or selection, e.g., for speed of protein translation and correct folding. However, this codon bias can also be affected by a general selection at the amino acid level due to differences in the acceptance of the loss and generation of these codons. To assess the importance of this effect, we constructed a mutation-selection model model, in which we generated almost 90,000 stationary nucleotide distributions produced by mutational processes and applied a selection based on differences in physicochemical properties of amino acids. Under these conditions, we calculated the usage of fourfold degenerated (4FD) codons and compared it with the usage characteristic of the pure mutations. We considered both the standard genetic code (SGC) and alternative genetic codes (AGCs). The analyses showed that a majority of AGCs produced a greater 4FD codon bias than the SGC. The mutations producing more thymine or adenine than guanine and cytosine increased the differences in usage. On the other hand, the mutational pressures generating a lot of cytosine or guanine with a low content of adenine and thymine decreased this bias because the nucleotide content of most 4FD codons stayed in the compositional equilibrium with these pressures. The comparison of the theoretical results with those for real protein coding sequences showed that the influence of selection at the amino acid level on the synonymous codon usage cannot be neglected. The analyses indicate that the effect of amino acid selection cannot be disregarded and that it can interfere with other selection factors influencing codon usage, especially in AT-rich genomes, in which AGCs are usually used.

The DEAD-box protein Dbp6 is an ATPase and RNA annealase interacting with the peptidyl transferase center (PTC) of the ribosome

Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA-RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that in vitro, Dbp6 shows ATPase as well as annealing and clamping activities negatively regulated by ATP. Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5' domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.

The Influence of the Nucleotide Composition of Genes and Gene Regulatory Elements on the Efficiency of Protein Expression in Escherichia coli

Recombinant proteins expressed in Escherichia coli are widely used in biochemical research and industrial processes. At the same time, achieving higher protein expression levels and correct protein folding still remains the key problem, since optimization of nutrient media, growth conditions, and methods for induction of protein synthesis do not always lead to the desired result. Often, low protein expression is determined by the sequences of the expressed genes and their regulatory regions. The genetic code is degenerated; 18 out of 20 amino acids are encoded by more than one codon. Choosing between synonymous codons in the coding sequence can significantly affect the level of protein expression and protein folding due to the influence of the gene nucleotide composition on the probability of formation of secondary mRNA structures that affect the ribosome binding at the translation initiation phase, as well as the ribosome movement along the mRNA during elongation, which, in turn, influences the mRNA degradation and the folding of the nascent protein. The nucleotide composition of the mRNA untranslated regions, in particular the promoter and Shine-Dalgarno sequences, also affects the efficiency of mRNA transcription, translation, and degradation. In this review, we describe the genetic principles that determine the efficiency of protein production in Escherichia coli.

Lectin Affinity Chromatography.

Glycosylation is a non-template-driven posttranslational modification during which linked-sugars and glycans are added to the nascent polypeptide. Over 70% of the eukaryotic proteome is thought to be glycosylated. It is now known that correct glycosylation is essential for the correct folding, solubility, stability, and immunogenicity of proteins. In this chapter, we describe the technique of lectin affinity chromatography (LAC), a procedure that has the ability to distinguish different glycans, which are attached to proteins or lipids, termed glycoproteins or glycolipids, respectively. This method utilizes different immobilized lectins that have affinity for specific sugar substrates, to separate a wide range of glycan-attached complexes (Ambrosi et al., Org Biomol Chem 3:1593-1608, 2005). To further enhance the specificity of LAC, a corresponding free sugar may be used to produce a specific elution. In general, the conditions under which lectin affinity chromatography operates are relatively mild resulting in good biological recoveries of the glycoproteins.

A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli

Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.

АЛГОРИТМ РЕФЕРУВАННЯ ПУБЛІЦИСТИЧНОГО ТЕКСТУ

The proposed linguistic didactic study is devoted to the problem of forming the skills and abilities of philology bachelors in the analytical and synthetic processing of journalistic Ukrainian-language text and the creation of their own German-language excerpt. The article provides methodological recommendations for teaching bilingual excerpting of a Ukrainian-language journalistic text in German for students of higher education of the first bachelor's level in the field of knowledge 03 Humanities, specialty 035 Philology, under the educational and professional program «Language and Literature (German / English). Translation». A four-year-tested algorithm for bilingual abstracting of a magazine or newspaper article is proposed, which practically helps students of the Faculty of Foreign Philology master the technology of working with modern journalistic text at an advanced stage, i.e. level B2 according to the All-European Recommendations on language education. The correct automated observance of all steps of the algorithm (familiarization with the journalistic text, careful reading of the article, translation recoding of the excerpt, design of the written excerpt, self-control of the written excerpt, editing of the text of the excerpt) contributes to the correct semantic folding and adequate linguistic compression of the source text, which contributes to the optimization of the educational process under time to study German as a primary and second foreign language. The peculiarities of the structure of the excerpt as a specific secondary document are given: the exordium (bibliographic description of the primary document), the abstract part itself (the basis of the excerpt, that is, the information obtained by analytically and synthetically processing the content of the primary text) and the conclusion (addition). Creating an excerpt according to the proposed effective algorithm significantly saves time and effort of higher education students and, accordingly, increases their motivation to study. The difficulties that accompanied higher education applicants during the compilation of an excerpt were analyzed.

Ubiquitin-like protein MNSFβ regulates glycolysis and promotes cell proliferation with HSC70 assistance

Monoclonal non-specific suppressor factor β (MNSFβ) is a universally expressed ubiquitin-like protein that has multiple biological functions. MNSFβ modifies its target molecules through covalent conjugation. Most recently, we identified a molecular chaperone, HSC70, that facilitates the stabilization of aggregable MNSFβ. In the current study, we determined the role of HSC70 in stabilizing unstable MNSFβ. HSC70 promoted the correct folding of MNSFβ both in vitro and in vivo. We also examined the regulatory function of MNSFβ in cell proliferation and glycolysis. MNSFβ siRNA and HSC70 siRNA treatment attenuated lactate release from Raw264.7 macrophage-like cells. MNSFβ siRNA inhibited glucose uptake in Raw264.7 cells. We found that glucose transporter 1 (GLUT1) is an important membrane protein involved in the regulatory function of MNSFβ during glycolysis. MNSFβ siRNA inhibited the increased GLUT1 expression in LPS-stimulated cells, suggesting that MNSFβ controls the inflammatory response through GLUT1 regulation. We identified several important molecules, including lactate dehydrogenase A, which are regulated by MNSFβ and involved in glucose metabolism. Here we firstly report that MNSFβ regulates glycolysis and promotes cell proliferation.

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

Iron–sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe–S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe–4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron–sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered “SufFeScient” derivative of BL21(DE3). Our results show that co-expression of a Fe–S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe–S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe–S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe–S cluster maturation and points out the pitfalls of chemical reconstitution.Graphical abstract

Epichaperome Inhibition Targets Kinase and TP53 Mutant Therapy Resistant AML

The BCL-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, improves outcomes for many patients with AML and is now considered standard of care for patients older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months; 2.5 months after relapse. Mutations of FLT3, PTPN11, and RAS, leading to constitutive activation of cell survival signaling, and mutations of TP53 have been identified as key determinants of low response rates, early relapse and death. Mutant TP53 cells are also characterized by activation of signaling kinases. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. Epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, epichaperome inhibition has the potential of eliminating resistant subclones before they become dominant and cause relapse. In contrast to other HSP90 purine analog inhibitors, PU-H71(zelavespib) is a competitive inhibitor specific for the conformationally changed ATP binding site of HSP90 when incorporated into epichaperomes. We here investigate the therapeutic potential of targeting epichaperomes with PU-H71 in TP53 and oncogenic kinase mutant AML. Western blot analysis showed increased HSP90 and several signaling proteins such as STAT3 and c-Myc in TP53-knockout and mutant Molm13 cells generated by CRISPR/cas-9, compared to isogeneic wild-type (WT) controls. Using fluorochrome-labelled PU-H71 and flow cytometry, we demonstrated the presence of epichaperome in AML and AML stem/progenitor cells with TP53 and/or RAS mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively killed AML cells, including those with acquired resistance to BH3 mimetics and those with various FLT3 mutants. PU-H71 targets AML cells and stem/progenitor cells with TP53 mutations, and prolonged survival of TP53-mutant AML xenograft mice with minimal effects on mouse bone marrow LSK cells and hematopoiesis. PU-H71 increased the expression of pro-apoptotic BIM and the efficacy of VEN in AML cells and AML stem/progenitor cells with TP53 and/or RAS mutations (combination index < 1). Importantly, in a 1,000: 1 mixture of TP53-WT and TP53-R248W mutant isogenic Molm13 cells, MDM2 inhibitor nutlin3a selectively killed TP53-WT but enriched TP53-mutant Molm13 cells, as expected, and VEN treatment also favored the outgrowth of TP53-mutant cells. However, PU-H71 effectively killed TP53-WT and mutant cells (Fig. 1): 10 million TP53-WT cells were reduced to 335 cells, and 10,000 TP53-mutant cells to 26. The activity of PU-H71 against both TP53-WT and mutant AML cells was further enhanced by VEN in vivo in a xenograft model of mixed (10:1) TP53-WT and mutant Molm13 cells. Our data support the concept that the epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring TP53 and/or oncogenic kinase mutations. Inhibition of epichaperomes by PU-H71 targets AML cells/stem/progenitor cells, enhances VEN activity, and prevents the outgrowth of nutlin- or VEN-resistant TP53-mutant AML cells. This concept has entered clinical evaluation in MPN-derived secondary AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

4-phenylbutyric acid-Identity crisis; can it act as a translation inhibitor?

Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4-phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore-it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases.

Emulation of the structure of the Saposin protein fold by a lung surfactant peptide construct of surfactant Protein B.

The three-dimensional structure of the synthetic lung Surfactant Protein B Peptide Super Mini-B was determined using an integrative experimental approach, including mass spectrometry and isotope enhanced Fourier-transform infrared (FTIR) spectroscopy. Mass spectral analysis of the peptide, oxidized by solvent assisted region-specific disulfide formation, confirmed that the correct folding and disulfide pairing could be facilitated using two different oxidative structure-promoting solvent systems. Residue specific analysis by isotope enhanced FTIR indicated that the N-terminal and C-terminal domains have well defined α-helical amino acid sequences. Using these experimentally derived measures of distance constraints and disulfide connectivity, the ensemble was further refined with molecular dynamics to provide a medium resolution, residue-specific structure for the peptide construct in a simulated synthetic lung surfactant lipid multilayer environment. The disulfide connectivity combined with the α-helical elements stabilize the peptide conformationally to form a helical hairpin structure that resembles critical elements of the Saposin protein fold of the predicted full-length Surfactant Protein B structure.

Insights into the structure and function of the histidine kinase ComP from Bacillus amyloliquefaciens based on molecular modeling.

The ComPA two-component signal transduction system (TCS) is essential in Bacillus spp. However, the molecular mechanism of the histidine kinase ComP remains unclear. Here, we predicted the structure of ComP from Bacillus amyloliquefaciens Q-426 (BaComP) using an artificial intelligence approach, analyzed the structural characteristics based on the molecular docking results and compared homologous proteins, and then investigated the biochemical properties of BaComP. We obtained a truncated ComPS protein with high purity and correct folding in solution based on the predicted structures. The expression and purification of BaComP proteins suggested that the subdomains in the cytoplasmic region influenced the expression and stability of the recombinant proteins. ComPS is a bifunctional enzyme that exhibits the activity of both histidine kinase and phosphotransferase. We found that His571 played an obligatory role in the autophosphorylation of BaComP based on the analysis of the structures and mutagenesis studies. The molecular docking results suggested that the HATPase_c domain contained an ATP-binding pocket, and the ATP molecule was coordinated by eight conserved residues from the N, G1, and G2 boxes. Our study provides novel insight into the histidine kinase BaComP and its homologous proteins.

Dry Molten Globule-Like Intermediates in Protein Folding, Function, and Disease.

The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding-unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.

Conformational Analysis of the Loop-to-Helix Transition of the α-Helix3 Plastic Region in the N-Terminal Domain of Human Hsp90α by a Computational Biochemistry Approach.

Hsp90 is a chaperone protein aiding in correct protein folding and attractive for drug discovery. The structure of human Hsp90α N-terminal domain (NTD) is intriguing since the α-helix3 region of the ATP-binding site in the NTD plastically changes its conformation, i.e., loop-out, loop-in, and helical conformations, according to the bound inhibitor type. The plastic region structure is known to influence the mode of inhibition-inhibitors bound to a helix have a longer residence time in the complex, which is a factor of in vivo-active drugs, compared with loop binders. In this study, we analyzed the loop-to-helix transition of the plastic region through binding of a helix binder by a computational biochemistry approach. To generate the helical transition from the loop, the resorcinol inhibitor C1 complexed with a loop-in structure was alchemically transformed to the C10 inhibitor, which is known as a helix binder. The loop in the C1 complex possesses Leu107 tightly binding to the hydrophobic subpocket, considered as a key residue for the plasticity. From 10 × 1 μs simulations after the alchemical transformation, the helical transition was observed with a 29% success rate. Conformational analysis of the simulations identified residues possibly associated with the helical transition. The implementation of additional simulations (dihedral-constrained and in silico mutant simulations) led to a statistically significant increase in the transition success rate to 78%, as observed in Asn105 psi-constrained simulation. Therefore, we concluded that the Asn105 psi dihedral angle is most likely involved in the helical transition by a change of the dihedral angle to gauche-negative.

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases.

β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers – they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Exploring the effect of tethered domains on the folding of Grb2 protein

Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding.

Relationship between heat shock proteins and cellular resistance to drugs and ageing.

Ageing is a multifactorial degenerative process which causes a decrease in the cellular capacity for repair and adaptation to external stressors. In this way, it is important to maintain the proper balance of the proteome. Heat shock proteins (HSP) will intervene in this balance, which are responsible for the correct assembly, folding and translocation of other proteins when cells are subjected to stressors. This type of protein is overexpressed in human tumor cells, while its deficit, both in function and quantity, contributes to ageing processes. The present work aims to analyze the response of cells from studies carried out in normal and tumor cells that are subjected to stressors. A PubMed search was performed using the keywords "cell ageing, cell longevity, resistance, HSP, heat shock proteins, thermal shock proteins". This search generated 212 articles. Subsequently, a series of inclusion and exclusion criteria were applied to select the articles of interest to be evaluated. Normal cells subjected to external stressors at low doses increase the number of HSP, causing them to become more resistant. In addition, tumor cells expressing high levels of HSP show greater resistance to treatment and increased cell replication. HSP intervene in the cellular resistance of both normal and tumor cells. In the case of normal cells, the increase in HSP levels makes them respond effectively to an external stressor, increasing their resistance and not causing cell death. In the case of tumor cells, there is an increase in resistance to treatment.

Inhibition of human peptide deformylase by actinonin sensitizes glioblastoma cells to temozolomide chemotherapy

Glioblastoma multiforme (GBM) is a common intracranial primary tumor of the central nervous system with high malignancy, poor prognosis, and short survival. Studies have shown that mitochondrial energy metabolism plays an important role in GBM chemotherapy resistance, suggesting that interrupting mitochondrial oxidative phosphorylation (OXPHOS) may improve GBM treatment. Human peptide deformylase (HsPDF) is a mitochondrial deformylase that removes the formylated methionine from the N-terminus of proteins encoded by mitochondrial DNA (mtDNA), thereby contributing to correct protein folding and participating in the assembly of the electron respiratory chain complex. In this study, we found that the expression of mtDNA-encoded proteins was significantly downregulated after treatment of GBM cells U87MG and LN229 with the HsPDF inhibitor, actinonin. In combination with temozolomide, a preferred chemotherapeutic medicine for GBM, the OXPHOS level decreased, mitochondrial protein homeostasis was unbalanced, mitochondrial fission increased, and the integrated stress response was activated to promote mitochondrial apoptosis. These findings suggest that HsPDF inhibition is an important strategy for overcoming chemoresistance of GBM cells.