Interplay between myotubularins and Ca2+ homeostasis.

The myotubularin family, encompassing myotubularin 1 (MTM1) and 14 myotubularin-related proteins (MTMRs), represents a conserved group of phosphatases featuring a protein tyrosine phosphatase domain. Nine members are characterized by an active phosphatase domain C(X)5R, dephosphorylating the D3 position of PtdIns(3)P and PtdIns(3,5)P2. Mutations in myotubularin genes result in human myopathies, and several neuropathies including X-linked myotubular myopathy and Charcot-Marie-Tooth type 4B. MTM1, MTMR6 and MTMR14 also contribute to Ca2+ signaling and Ca2+ homeostasis that play a key role in many MTM-dependent myopathies and neuropathies. Here we explore the evolving roles of MTM1/MTMRs, unveiling their influence on critical aspects of Ca2+ signaling pathways.

Sense and anti-sense: Role of FAM83A and FAM83A-AS1 in Wnt, EGFR, PI3K, EMT pathways and tumor progression.

An increasing number of studies have shown that FAM83A, a member of the family with sequence similarity 83 (FAM83), which consists of eight members, is a key tumor therapeutic target involved in multiple signaling pathways. It has been reported that FAM83A plays essential roles in the regulation of Wnt/ß-catenin, EGFR, MAPK, EMT, and other signaling pathways and physiological processes in models of pancreatic cancer, lung cancer, breast cancer, and other malignant tumors. Moreover, the expression of FAM83A could be significantly affected by multiple noncoding RNAs that are dysregulated in malignant tumors, the dysregulation of which is essential for the malignant process. Among these noncoding RNAs, the most noteworthy is the antisense long noncoding (Lnc) RNA of FAM83A itself (FAM83A-AS1), indicating an outstanding synergistic carcinogenic effect between FAM83A and FAM83A-AS1. In the present study, the specific mechanisms by which FAM83A and FAM83A-AS1 cofunction in the Wnt/ß-catenin and EGFR signaling pathways were reviewed in detail, which will guide subsequent research. We also described the applications of FAM83A and FAM83A-AS1 in tumor therapy and provided a certain theoretical basis for subsequent drug target development and combination therapy strategies.

The TRPV6 Calcium Channel and Its Relationship with Cancer.

Transient receptor potential vanilloid-6 (TRPV6) is a cation channel belonging to the TRP superfamily, specifically the vanilloid subfamily, and is the sixth member of this subfamily. Its presence in the body is primarily limited to the skin, ovaries, kidney, testes, and digestive tract epithelium. The body maintains calcium homeostasis using the TRPV6 channel, which has a greater calcium selectivity than the other TRP channels. Several pieces of evidence suggest that it is upregulated in the advanced stages of thyroid, ovarian, breast, colon, and prostate cancers. The function of TRPV6 in regulating calcium signaling in cancer will be covered in this review, along with its potential applications as a cancer treatment target.

Elevated external temperature affects cell ultrastructure and heat shock proteins (HSPs) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020.

Increasing temperature influences the habitats of various organisms, including microscopic invertebrates. To gain insight into temperature-dependent changes in tardigrades, we isolated storage cells exposed to various temperatures and conducted biochemical and ultrastructural analysis in active and tun-state Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020. The abundance of heat shock proteins (HSPs) and ultrastructure of the storage cells were examined at different temperatures (20 °C, 30 °C, 35 °C, 37 °C, 40 °C, and 42 °C) in storage cells isolated from active specimens of Pam. experimentalis. In the active animals, upon increase in external temperature, we observed an increase in the levels of HSPs (HSP27, HSP60, and HSP70). Furthermore, the number of ultrastructural changes in storage cells increased with increasing temperature. Cellular organelles, such as mitochondria and the rough endoplasmic reticulum, gradually degenerated. At 42 °C, cell death occurred by necrosis. Apart from the higher electron density of the karyoplasm and the accumulation of electron-dense material in some mitochondria (at 42 °C), almost no changes were observed in the ultrastructure of tun storage cells exposed to different temperatures. We concluded that desiccated (tun-state) are resistant to high temperatures, but not active tardigrades (survival rates of tuns after 24 h of rehydration: 93.3% at 20 °C, 60.0% at 35 °C, 33.3% at 37 °C, 33.3% at 40 °C, and 20.0% at 42 °C).

ILKAP Promotes the Metastasis of Hepatocellular Carcinoma Cells by Inhibiting ß-Catenin Degradation and Enhancing the WNT Signaling Pathway.

The incidence of Hepatocellular carcinoma (HCC) and HCC-related deaths have remarkably increased over the recent decades. It has been reported that ß-catenin activation can be frequently observed in HCC cases. This study identified the integrin-linked kinase-associated phosphatase (ILKAP) as a novel ß-catenin-interacting protein. ILKAP is localized both in the nucleus and cytoplasm and regulates the WNT pathway in different ways. First, it is demonstrated that ILKAP activates the WNT pathway in HCC cells by increasing the protein level of ß-catenin and other proteins associated with the WNT signaling, such as c-Myc and CyclinD1. Next, it is shown that ILKAP promotes the metastasis of HCC both in vitro and in vivo in a zebrafish xenograft model. It is also found that ILKAP dephosphorylates the GSK3ß and CK1, contributing to the reduced ubiquitination of ß-catenin. Furthermore, it is identified that ILKAP functions by mediating binding between TCF4 and ß-catenin to enhance expression of WNT target genes. Taken together, the study demonstrates a critical function of ILKAP in metastasis of HCC, since ILKAP is crucial for the activation of the WNT pathway via stabilization of ß-catenin and increased binding between TCF4 and ß-catenin.

Calreticulin Regulates SARS-CoV-2 Spike Protein Turnover and Modulates SARS-CoV-2 Infectivity.

Cardiovascular complications are major clinical hallmarks of acute and post-acute coronavirus disease 2019 (COVID-19). However, the mechanistic details of SARS-CoV-2 infectivity of endothelial cells remain largely unknown. Here, we demonstrate that the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein shares a similarity with the proline-rich binding ena/VASP homology (EVH1) domain and identified the endoplasmic reticulum (ER) resident calreticulin (CALR) as an S-RBD interacting protein. Our biochemical analysis showed that CALR, via its proline-rich (P) domain, interacts with S-RBD and modulates proteostasis of the S protein. Treatment of cells with the proteasomal inhibitor bortezomib increased the expression of the S protein independent of CALR, whereas the lysosomal/autophagy inhibitor bafilomycin 1A, which interferes with the acidification of lysosome, selectively augmented the S protein levels in a CALR-dependent manner. More importantly, the shRNA-mediated knockdown of CALR increased SARS-CoV-2 infection and impaired calcium homeostasis of human endothelial cells. This study provides new insight into the infectivity of SARS-CoV-2, calcium hemostasis, and the role of CALR in the ER-lysosome-dependent proteolysis of the spike protein, which could be associated with cardiovascular complications in COVID-19 patients.

RUNDC1 inhibits autolysosome formation and survival of zebrafish via clasping ATG14-STX17-SNAP29 complex.

Autophagy serves as a pro-survival mechanism for a cell or a whole organism to cope with nutrient stress. Our understanding of the molecular regulation of this fusion event remains incomplete. Here, we identified RUNDC1 as a novel ATG14-interacting protein, which is highly conserved across vertebrates, including zebrafish and humans. By gain and loss of function studies, we demonstrate that RUNDC1 negatively modulates autophagy by blocking fusion between autophagosomes and lysosomes via inhibiting the assembly of the STX17-SNAP29-VAMP8 complex both in human cells and the zebrafish model. Moreover, RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation. This also prevents VAMP8 from binding to STX17-SNAP29. We further identified that phosphorylation of RUNDC1 Ser379 is crucial to inhibit the assembly of the STX17-SNAP29-VAMP8 complex via promoting ATG14 homo-oligomerization. In line with our findings, RunDC1 is crucial for zebrafish in their response to nutrient-deficient conditions. Taken together, our findings demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes by clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding.

Phosphorylated PTTG1 switches its subcellular distribution and promotes ß-catenin stabilization and subsequent transcription activity.

The Wnt/ß-catenin signaling is usually abnormally activated in hepatocellular carcinoma (HCC), and pituitary tumor-transforming gene 1 (PTTG1) has been found to be highly expressed in HCC. However, the specific mechanism of PTTG1 pathogenesis remains poorly understood. Here, we found that PTTG1 is a bona fide ß-catenin binding protein. PTTG1 positively regulates Wnt/ß-catenin signaling by inhibiting the destruction complex assembly, promoting ß-catenin stabilization and subsequent nuclear localization. Moreover, the subcellular distribution of PTTG1 was regulated by its phosphorylation status. Among them, PP2A induced PTTG1 dephosphorylation at Ser165/171 residues and prevented PTTG1 translocation into the nucleus, but these effects were effectively reversed by PP2A inhibitor okadaic acid (OA). Interestingly, we found that PTTG1 decreased Ser9 phosphorylation-inactivation of GSK3ß by competitively binding to PP2A with GSK3ß, indirectly leading to cytoplasmic ß-catenin stabilization. Finally, PTTG1 was highly expressed in HCC and associated with poor patient prognosis. PTTG1 could promote the proliferative and metastasis of HCC cells. Overall, our results indicated that PTTG1 plays a crucial role in stabilizing ß-catenin and facilitating its nuclear accumulation, leading to aberrant activation of Wnt/ß-catenin signaling and providing a feasible therapeutic target for human HCC.

Calreticulin: Endoplasmic reticulum Ca2+ gatekeeper.

Endoplasmic reticulum (ER) luminal Ca2+ is vital for the function of the ER and regulates many cellular processes. Calreticulin is a highly conserved, ER-resident Ca2+ binding protein and lectin-like chaperone. Over four decades of studying calreticulin demonstrate that this protein plays a crucial role in maintaining Ca2+ supply under different physiological conditions, in managing access to Ca2+ and how Ca2+ is used depending on the environmental events and in making sure that Ca2+ is not misused. Calreticulin plays a role of ER luminal Ca2+ sensor to manage Ca2+ -dependent ER luminal events including maintaining interaction with its partners, Ca2+ handling molecules, substrates and stress sensors. The protein is strategically positioned in the lumen of the ER from where the protein manages access to and distribution of Ca2+ for many cellular Ca2+ -signalling events. The importance of calreticulin Ca2+ pool extends beyond the ER and includes influence of cellular processes involved in many aspects of cellular pathophysiology. Abnormal handling of the ER Ca2+ contributes to many pathologies from heart failure to neurodegeneration and metabolic diseases.

Therapeutic strategies targeting AMPK-dependent autophagy in cancer cells.

Macroautophagy is a health-modifying process of engulfing misfolded or aggregated proteins or damaged organelles, coating these proteins or organelles into vesicles, fusion of vesicles with lysosomes to form autophagic lysosomes, and degradation of the encapsulated contents. It is also a self-rescue strategy in response to harsh environments and plays an essential role in cancer cells. AMP-activated protein kinase (AMPK) is the central pathway that regulates autophagy initiation and autophagosome formation by phosphorylating targets such as mTORC1 and unc-51 like activating kinase 1 (ULK1). AMPK is an evolutionarily conserved serine/threonine protein kinase that acts as an energy sensor in cells and regulates various metabolic processes, including those involved in cancer. The regulatory network of AMPK is complicated and can be regulated by multiple upstream factors, such as LKB1, AKT, PPAR, SIRT1, or noncoding RNAs. Currently, AMPK is being investigated as a novel target for anticancer therapies based on its role in macroautophagy regulation. Herein, we review the effects of AMPK-dependent autophagy on tumor cell survival and treatment strategies targeting AMPK.

Membrane Curvature: The Inseparable Companion of Autophagy.

Autophagy is a highly conserved recycling process of eukaryotic cells that degrades protein aggregates or damaged organelles with the participation of autophagy-related proteins. Membrane bending is a key step in autophagosome membrane formation and nucleation. A variety of autophagy-related proteins (ATGs) are needed to sense and generate membrane curvature, which then complete the membrane remodeling process. The Atg1 complex, Atg2-Atg18 complex, Vps34 complex, Atg12-Atg5 conjugation system, Atg8-phosphatidylethanolamine conjugation system, and transmembrane protein Atg9 promote the production of autophagosomal membranes directly or indirectly through their specific structures to alter membrane curvature. There are three common mechanisms to explain the change in membrane curvature. For example, the BAR domain of Bif-1 senses and tethers Atg9 vesicles to change the membrane curvature of the isolation membrane (IM), and the Atg9 vesicles are reported as a source of the IM in the autophagy process. The amphiphilic helix of Bif-1 inserts directly into the phospholipid bilayer, causing membrane asymmetry, and thus changing the membrane curvature of the IM. Atg2 forms a pathway for lipid transport from the endoplasmic reticulum to the IM, and this pathway also contributes to the formation of the IM. In this review, we introduce the phenomena and causes of membrane curvature changes in the process of macroautophagy, and the mechanisms of ATGs in membrane curvature and autophagosome membrane formation.

Interplay between calcium and endoplasmic reticulum stress.

Cellular homeostasis is crucial for the healthy functioning of the organism. Disruption of cellular homeostasis activates endoplasmic reticulum (ER) stress coping responses including the unfolded protein response (UPR). There are three ER resident stress sensors responsible for UPR activation - IRE1α, PERK and ATF6. Ca2+ signaling plays an important role in stress responses including the UPR and the ER is the main Ca2+ storage organelle and a source of Ca2+ for cell signaling. The ER contains many proteins involved in Ca2+ import/export/ storage, Ca2+ movement between different cellular organelles and ER Ca2+ stores refilling. Here we focus on selected aspects of ER Ca2+ homeostasis and its role in activation of the ER stress coping responses.

Computational Analysis and Experimental Testing of the Molecular Mode of Action of Gatastatin and Its Derivatives.

Given its critical role in cell mitosis, the tubulin γ chain represents a viable chemotherapeutic target to solve the specificity issues associated with targeting α and ß tubulin. Since γ tubulin is overexpressed in glioblastoma multiforme (GBM) and some breast lesions, the glaziovianin A derivative gatastatin, presented as a γ-tubulin-specific inhibitor, could yield a successful therapeutic strategy. The present work aims to identify the binding sites and modes of gatastatin and its derivatives through molecular-docking simulations. Computational binding free energy predictions were compared to experimental microscale thermophoresis assay results. The computational simulations did not reveal a strong preference toward γ tubulin, suggesting that further derivatization may be needed to increase its specificity.

B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with ß-catenin.

Abnormal activation of Wnt/ß-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to ß-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-ß-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/ß-catenin-mediated transcription through promoting ß-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/ß-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-ß-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer.

Calnexin, More Than Just a Molecular Chaperone.

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca2+-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.

Cisplatin Toxicity Is Mediated by Direct Binding to Toll-Like Receptor 4 through a Mechanism That Is Distinct from Metal Allergens.

Cisplatin is an effective chemotherapeutic agent, yet its use is limited by several adverse drug reactions, known as cisplatin-induced toxicities (CITs). We recently demonstrated that cisplatin could elicit proinflammatory responses associated with CITs through Toll-like receptor 4 (TLR4). TLR4 is best recognized for binding bacterial lipopolysaccharide (LPS) via its coreceptor, MD-2. TLR4 is also proposed to directly bind transition metals, such as nickel. Little is known about the nature of the cisplatin-TLR4 interaction. Here, we show that soluble TLR4 was capable of blocking cisplatin-induced, but not LPS-induced, TLR4 activation. Cisplatin and nickel, but not LPS, were able to directly bind soluble TLR4 in a microscale thermophoresis binding assay. Interestingly, TLR4 histidine variants that abolish nickel binding reduced, but did not eliminate, cisplatin-induced TLR4 activation. This was corroborated by binding data that showed cisplatin, but not nickel, could directly bind mouse TLR4 that lacks these histidine residues. Altogether, our findings suggest that TLR4 can directly bind cisplatin in a manner that is enhanced by, but not dependent on, histidine residues that facilitate binding to transition metals. SIGNIFICANCE STATEMENT: This work describes how the xenobiotic cisplatin interacts with Toll-like receptor 4 (TLR4) to initiate proinflammatory signaling that underlies cisplatin toxicities, which are severe adverse outcomes in cisplatin treatment. Here, this study provides a mechanistic bridge between cisplatin extracellular interactions with TLR4 and previous observations that genetic and chemical inhibition of TLR4 mitigates cisplatin-induced toxicity.

Loss of the fructose transporter SLC2A5 inhibits cancer cell migration.

Metastasis is the primary cause of cancer patient death and the elevation of SLC2A5 gene expression is often observed in metastatic cancer cells. Here we evaluated the importance of SLC2A5 in cancer cell motility by silencing its gene. We discovered that CRISPR/Cas9-mediated inactivation of the SLC2A5 gene inhibited cancer cell proliferation and migration in vitro as well as metastases in vivo in several animal models. Moreover, SLC2A5-attenuated cancer cells exhibited dramatic alterations in mitochondrial architecture and localization, uncovering the importance of SLC2A5 in directing mitochondrial function for cancer cell motility and migration. The direct association of increased abundance of SLC2A5 in cancer cells with metastatic risk in several types of cancers identifies SLC2A5 as an important therapeutic target to reduce or prevent cancer metastasis.

Computational Prediction and Experimental Validation of the Unique Molecular Mode of Action of Scoulerine.

Scoulerine is a natural compound that is known to bind to tubulin and has anti-mitotic properties demonstrated in various cancer cells. Its molecular mode of action has not been precisely known. In this work, we perform computational prediction and experimental validation of the mode of action of scoulerine. Based on the existing data in the Protein Data Bank (PDB) and using homology modeling, we create human tubulin structures corresponding to both free tubulin dimers and tubulin in a microtubule. We then perform docking of the optimized structure of scoulerine and find the highest affinity binding sites located in both the free tubulin and in a microtubule. We conclude that binding in the vicinity of the colchicine binding site and near the laulimalide binding site are the most likely locations for scoulerine interacting with tubulin. Thermophoresis assays using scoulerine and tubulin in both free and polymerized form confirm these computational predictions. We conclude that scoulerine exhibits a unique property of a dual mode of action with both microtubule stabilization and tubulin polymerization inhibition, both of which have similar affinity values.

The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy.

Transient receptor potential melastatin 8 (TRPM8) functions as a Ca2+-permeable channel in the plasma membrane (PM). Dysfunction of TRPM8 is associated with human pancreatic cancer and several other diseases in clinical patients, but the underlying mechanisms are unclear. Here, we found that lymphocyte-specific protein tyrosine kinase (LCK) directly interacts with TRPM8 and potentiates TRPM8 phosphorylation at Y1022. LCK positively regulated channel function characterized by increased TRPM8 current densities by enhancing TRPM8 multimerization. Furthermore, 14-3-3ζ interacted with TRPM8 and positively modulated channel multimerization. LCK significantly enhanced the binding of 14-3-3ζ and TRPM8, whereas mutant TRPM8-Y1022F impaired TRPM8 multimerization and the binding of TRPM8 and 14-3-3ζ. Knockdown of 14-3-3ζ impaired the regulation of TRPM8 multimerization by LCK. In addition, TRPM8 phosphotyrosine at Y1022 feedback regulated LCK activity by inhibiting Tyr505 phosphorylation and modulating LCK ubiquitination. Finally, we revealed the importance of TRPM8 phosphorylation at Y1022 in the proliferation, migration, and tumorigenesis of pancreatic cancer cells. Our findings demonstrate that the LCK-14-3-3ζ-TRPM8 axis for regulates TRPM8 assembly, channel function, and LCK activity and maybe provide potential therapeutic targets for pancreatic cancer.

Calreticulin and the Heart.

Calreticulin is an endoplasmic Ca2+ binding protein and molecular chaperone. As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.