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Pancreatic β‑cells are the only cells that synthesize insulin to regulate blood glucose levels. Various conditions can affect the mass of pancreatic β‑cells and decrease insulin levels. Diabetes mellitus is a disease characterized by insulin resistance and chronic hyperglycemia, mainly due to the loss of pancreatic β‑cells caused by an increase in the rate of apoptosis. Additionally, hyperglycemia has a toxic effect on β‑cells. Although the precise mechanism of glucotoxicity is not fully understood, several mechanisms have been proposed. The most prominent changes are increases in reactive oxygen species, the loss of mitochondrial membrane potential and the activation of the intrinsic pathway of apoptosis due to p53. The present review analyzed the location of p53 in the cytoplasm, mitochondria and nucleus in terms of post‑translational modifications, including phosphorylation, O‑GlcNAcylation and poly‑ADP‑ribosylation, under hyperglycemic conditions. These modifications protect p53 from degradation by the proteasome and, in turn, enable it to regulate the intrinsic pathway of apoptosis through the regulation of anti‑apoptotic and pro‑apoptotic elements. Degradation of p53 occurs in the proteasome and depends on its ubiquitination by Mdm2. Understanding the mechanisms that activate the death of pancreatic β‑cells will allow the proposal of treatment alternatives to prevent the decrease in pancreatic β‑cells.

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme's structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI's susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging childhood cancer to treat, with limited therapeutic options and high relapse rates. This study explores deamidated triosephosphate isomerase (dTPI) as a novel therapeutic target. We hypothesized that selectively inhibiting dTPI could reduce T-ALL cell viability without affecting normal T lymphocytes. Computational modeling and recombinant enzyme assays revealed that disulfiram (DS) and curcumin (CU) selectively bind and inhibit dTPI activity without affecting the non-deamidated enzyme. At the cellular level, treatment with DS and CU significantly reduced Jurkat T-ALL cell viability and endogenous TPI enzymatic activity, with no effect on normal T lymphocytes, whereas the combination of sodium dichloroacetate (DCA) with DS or CU showed synergistic effects. Furthermore, we demonstrated that dTPI was present and accumulated only in Jurkat cells, confirming our hypothesis. Finally, flow cytometry confirmed apoptosis in Jurkat cells after treatment with DS and CU or their combination with DCA. These findings strongly suggest that targeting dTPI represents a promising and selective target for T-ALL therapy.

The classic enzymatic function of acetylcholinesterase (AChE) is the hydrolysis of acetylcholine (ACh) in the neuronal synapse. However, AChE is also present in nonneuronal cells such as lymphocytes. Various studies have proposed the participation of AChE in the development of cancer. The ACHE gene produces three mRNAs (T, H and R). AChE-T encodes amphiphilic monomers, dimers, tetramers (G 1 A , G 2 A and G 4 A ) and hydrophilic tetramers (G 4 H ). AChE-H encodes amphiphilic monomers and dimers (G 1 A and G 2 A ). AChE-R encodes a hydrophilic monomer (G 1 H ). The present study considered the differences in the mRNA expression (T, H and R) and protein levels of AChE, as well as the molecular forms of AChE, the glycosylation pattern and the enzymatic activity of AChE present in normal T lymphocytes and leukemic Jurkat E6-1 cells. The results revealed that AChE enzymatic activity was higher in normal T lymphocytes than in Jurkat cells. Normal T cells expressed AChE-H transcripts, whereas Jurkat cells expressed AChE-H and AChE-T. The molecular forms identified in normal T cells were G 2 A (5.2 S) and G 1 A (3.5 S), whereas those in Jurkat cells were G 2 A (5.2 S), G 1 A (3.5 S) and G 4 H (10.6S). AChE in Jurkat cells showed altered posttranslational maturation since a decrease in the incorporation of galactose and sialic acid into its structure was observed. In conclusion, the content and composition of AChE were altered in Jurkat cells compared with those in normal T lymphocytes. The present study opened new avenues for exploring the development of novel therapeutic strategies against T-cell leukemia and for identifying potential molecular targets for the early detection of this type of cancer., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Gómez-Olivares et al.)

Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi , but cellular enzyme inhibition has yet to be established. This study demonstrates that rabeprazole inhibits both cell viability and triosephosphate isomerase activity in T. cruzi epimastigotes. Our results show that rabeprazole has an IC 50 of 0.4 µM, which is 14.5 times more effective than benznidazole. Additionally, we observed increased levels of methyl-glyoxal and advanced glycation end products after the inhibition of cellular triosephosphate isomerase by rabeprazole. Finally, we demonstrate that the inactivation mechanisms of rabeprazole on triosephosphate isomerase of T. cruzi can be achieved through the derivatization of three of its four cysteine residues. These results indicate that rabeprazole is a promising candidate against American trypanosomiasis.

Cancer involves a series of diseases where cellular growth is not controlled. Cancer is a leading cause of death worldwide, and the burden of cancer incidence and mortality is rapidly growing, mainly in developing countries. Many drugs are currently used, from chemotherapeutic agents to immunotherapy, among others, along with organ transplantation. Treatments can cause severe side effects, including remission and progression of the disease with serious consequences. Increased glycolytic activity is characteristic of cancer cells. Triosephosphate isomerase is essential for net ATP production in the glycolytic pathway. Notably, some post-translational events have been described that occur in human triosephosphate isomerase in which functional and structural alterations are provoked. This is considered a window of opportunity, given the differences that may exist between cancer cells and their counterpart in normal cells concerning the glycolytic enzymes. Here, we provide elements that bring out the potential of triosephosphate isomerase, under post-translational modifications, to be considered an efficacious target for treating cancer.

Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia , arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular "chat" is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells.

Human triosephosphate isomerase (HsTIM) is a central glycolytic enzyme and is overexpressed in cancer cells with accelerated glycolysis. Triple-negative breast cancer is highly dependent on glycolysis and is typically treated with a combination of surgery, radiation therapy, and chemotherapy. Deamidated HsTIM was recently proposed as a druggable target. Although thiol-reactive drugs affect cell growth in deamidated HsTIM-complemented cells, the role of this protein as a selective target has not been demonstrated. To delve into the usefulness of deamidated HsTIM as a selective target, we assessed its natural accumulation in breast cancer cells. We found that deamidated HsTIM accumulates in breast cancer cells but not in noncancerous cells. The cancer cells are selectively programmed to undergo cell death with thiol-reactive drugs that induced the production of methylglyoxal (MGO) and advanced glycation-end products (AGEs). In vivo, a thiol-reactive drug effectively inhibits the growth of xenograft tumors with an underlying mechanism involving deamidated HsTIM. Our findings demonstrate the usefulness of deamidated HsTIM as target to develop new therapeutic strategies for the treatment of cancers and other pathologies in which this post translationally modified protein accumulates., (© 2022. The Author(s).)

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His 7 tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His 7 -TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His 7 -TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His 7 -TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)