Caspase-mediated AURKA cleavage at Asp132 is essential for paclitaxel to elicit cell apoptosis.

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.

Control of ENaC ubiquitination.

Ubiquitination influences the expression of the epithelial Na+ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and ßENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na+ could stimulate ubiquitination. Administration of amiloride to block Na+ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na+ produced by monensin in FRT cells or acute Na+ repletion in rats increased ubiquitination and decreased overall ENaC expression.NEW & NOTEWORTHY We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na+ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na+ can stimulate ENaC ubiquitination.

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation.

Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

DOT1L stimulates MYC/Mondo transcription factor activity by promoting its degradation cycle on chromatin.

The proto-oncogene c-MYC is a key representative of the MYC transcription factor network regulating growth and metabolism. MML-1 (Myc- and Mondo-like) is its homolog in C. elegans. The functional and molecular cooperation between c-MYC and H3 lysine 79 methyltransferase DOT1L was demonstrated in several human cancer types, and we have earlier discovered the connection between C. elegans MML-1 and DOT-1.1. Here, we demonstrate the critical role of DOT1L/DOT-1.1 in regulating c-MYC/MML-1 target genes genome-wide by ensuring the removal of "spent" transcription factors from chromatin by the nuclear proteasome. Moreover, we uncover a previously unrecognized proteolytic activity of DOT1L, which may facilitate c-MYC turnover. This new mechanism of c-MYC regulation by DOT1L may lead to the development of new approaches for cancer treatment.

Inhibition of human immunodeficiency virus (HIV-1) infectivity by expression of poorly or broadly neutralizing antibodies against Env in virus-producing cells.

The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein precursor (gp160) trimerizes, is modified by high-mannose glycans in the endoplasmic reticulum, and is transported via Golgi and non-Golgi secretory pathways to the infected cell surface. In the Golgi, gp160 is partially modified by complex carbohydrates and proteolytically cleaved to produce the mature functional Env trimer, which is preferentially incorporated into virions. Broadly neutralizing antibodies (bNAbs) generally recognize the cleaved Env trimer, whereas poorly neutralizing antibodies (pNAbs) bind the conformationally flexible gp160. We found that expression of bNAbs, pNAbs, or soluble/membrane forms of the receptor, CD4, in cells producing HIV-1 all decreased viral infectivity. Four patterns of co-expressed ligand:Env were observed: (i) ligands (CD4, soluble CD4-Ig, and some pNAbs) that specifically recognize the CD4-bound Env conformation resulted in uncleaved Envs lacking complex glycans that were not incorporated into virions; (ii) other pNAbs produced Envs with some complex carbohydrates and severe defects in cleavage, which were relieved by brefeldin A treatment; (iii) bNAbs that recognize gp160 as well as mature Envs resulted in Envs with some complex carbohydrates and moderate decreases in virion Env cleavage; and (iv) bNAbs that preferentially recognize mature Envs produced cleaved Envs with complex glycans in cells and on virions. The low infectivity observed upon co-expression of pNAbs or CD4 could be explained by disruption of Env trafficking, reducing the level of Env and/or increasing the fraction of uncleaved Env on virions. In addition to bNAb effects on virion Env cleavage, the secreted bNAbs neutralized the co-expressed viruses.IMPORTANCEThe Env trimers on the HIV-1 mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars, and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.

Temsavir Modulates HIV-1 Envelope Conformation by Decreasing Its Proteolytic Cleavage.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the ß20-ß21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC).

Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from Bacillus thuringiensis subsp. israelensis ISPC-12.

Cry11Aa is the most potent mosquito larvicidal protein of Bacillus thuringiensis subsp. israelensis (Bti). Development of resistance against insecticidal proteins including Cry11Aa is known but no field resistance was observed with Bti. The phenomenon of increasing resistance in insect pests necessitates the development of new strategies and techniques to enhance efficacy of insecticidal proteins. Recombinant technology offers better control over the molecule and allows modification of protein to achieve maximal effect against target pests. In this study, we standardised protocol for recombinant purification of Cry11Aa. Recombinant Cry11Aa found active against larvae of Aedes and Culex mosquito species and LC50 were estimated. Detailed biophysical characterization provides crucial insights into stability and in-vitro behaviour of the recombinant Cry11Aa. Moreover, trypsin hydrolysis doesn't improve overall toxicity of recombinant Cry11Aa. Proteolytic processing suggests domain I and II are more prone to proteolysis in comparison to domain III. Significance of structural features for proteolysis of Cry11Aa was observed after performing molecular dynamics simulations. Findings reported here are contributing significantly in method for purification, understanding in-vitro behaviour and proteolytic processing of Cry11Aa which could facilitate in efficient utilisation of Bti for insect pests and vectors control.

Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana.

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

The Dengue virus protease NS2B3 cleaves cyclic GMP-AMP synthase to suppress cGAS activation.

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human viruses that causes significant morbidity and mortality worldwide. To persist in the cell and consequently cause disease, DENV is evolved with mechanisms to suppress the induction of type I interferons by antagonizing cGAS-STING signaling. Using recombinant proteins and in vitro cleavage assays, we have shown that the DENV protease NS2B3 is capable of cleaving cGAS in the N-terminal region without disrupting the C-terminal catalytic center. This generates two major cleavage products: cleavage product N-terminal (CP-N) and cleavage product C-terminal (CP-C). We observed reduction in DNA-binding affinity of CP-C as compared to full-length cGAS. Reduction in DNA-binding affinity is also correlated with the decrease in enzymatic activity of CP-C. CP-N, on the other hand, has almost comparable DNA-binding ability as that of the full-length cGAS. In fact, CP-N competitively inhibits cyclic GMP-AMP production by both full-length cGAS and CP-C. We hypothesize that high DNA-binding affinity of CP-N enables it to sequester the DNA from CP-C and noncleaved full-length cGAS and thus reduces the rate of enzyme activation and cyclic GMP-AMP synthesis. Furthermore, we found that NS2B3 physically interacts with full-length cGAS and CP-C, laying the basis for their shuttling to and eventual degradation in the autophagosome. Overall, our study highlights a multifaceted and effective strategy by which an RNA virus antagonizes cGAS-STING signaling which may be useful for the design of antivirals targeting viral proteases.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

Improving bone morphogenetic protein (BMP) production in CHO cells through understanding of BMP synthesis, signaling and endocytosis.

Bone morphogenetic proteins (BMPs) are a group of growth factors with the clinical potential to regulate cartilage and bone formation. Functionally active mature recombinant human BMPs (rhBMPs), produced primarily in Chinese hamster ovary (CHO) cells for clinical applications, are considered difficult to express because they undergo maturation processes, signaling pathways, or endocytosis. Although BMPs are a family of proteins with similar mature domain sequence identities, their individual properties are diverse. Thus, understanding the properties of individual rhBMPs is essential to improve rhBMP production in CHO cells. In this review, we discuss various approaches to improve rhBMP production in CHO cells by understanding the overall maturation process, signaling pathways and endocytosis of individual rhBMPs.

Proteolytic cleavage of membrane proteins by membrane type-1 MMP regulates cancer malignant progression.

Strategies to develop cancer therapies using inhibitors that target matrix metalloproteinases (MMPs), particularly membrane type-1 MMP (MT1-MMP), have failed. This is predominantly attributed to the specificity of MMP inhibitors and numerous functions of MMPs; therefore, targeting substrates with such broad specificity can lead to off-target effects. Thus, new drug development for cancer therapeutics should focus on the ability of MT1-MMP to break down substrates, such as functional cell membrane proteins, to regulate the functions of these proteins that promote tumor malignancy. In this review, we discuss the mechanism by which proteolysis of cell surface proteins by MT1-MMP promotes progression of malignant tumor cells. In addition, we discuss the two protein fragments generated by limited cleavage of erythropoietin-producing hepatoma receptor tyrosine kinase A2 (EphA2-NF, -CF), which represent a promising basis for developing new cancer therapies and diagnostic techniques.

Anchorless risk or released benefit? An updated view on the ADAM10-mediated shedding of the prion protein.

The prion protein (PrP) is a broadly expressed glycoprotein linked with a multitude of (suggested) biological and pathological implications. Some of these roles seem to be due to constitutively generated proteolytic fragments of the protein. Among them is a soluble PrP form, which is released from the surface of neurons and other cell types by action of the metalloprotease ADAM10 in a process termed 'shedding'. The latter aspect is the focus of this review, which aims to provide a comprehensive overview on (i) the relevance of proteolytic processing in regulating cellular PrP functions, (ii) currently described involvement of shed PrP in neurodegenerative diseases (including prion diseases and Alzheimer's disease), (iii) shed PrP's expected roles in intercellular communication in many more (patho)physiological conditions (such as stroke, cancer or immune responses), (iv) and the need for improved research tools in respective (future) studies. Deeper mechanistic insight into roles played by PrP shedding and its resulting fragment may pave the way for improved diagnostics and future therapeutic approaches in diseases of the brain and beyond.

MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.

The involvement of the N-methyl-D-aspartate receptor (NMDAR), a glutamate-gated ion channel, in promoting the invasive growth of cancer cells is an area of ongoing investigation. Our previous findings revealed a physical interaction between NMDAR and MET, the hepatocyte growth factor (HGF) receptor. However, the molecular mechanisms underlying this NMDAR/MET interaction remain unclear. In this study, we demonstrate that the NMDAR2B subunit undergoes proteolytic processing, resulting in a low-molecular-weight form of 100 kDa. Interestingly, when the NMDAR2B and MET constructs were co-transfected, the full-size high-molecular-weight NMDAR2B form of 160 kDa was predominantly observed. The protection of NMDAR2B from cleavage was dependent on the kinase activity of MET. We provide the following evidence that MET opposes the autophagic lysosomal proteolysis of NMDAR2B: (i) MET decreased the protein levels of lysosomal cathepsins; (ii) treatment with either an inhibitor of autophagosome formation or the fusion of the autophagosome and lysosome elevated the proportion of the NMDAR2B protein's uncleaved form; (iii) a specific mTOR inhibitor hindered the anti-autophagic effect of MET. Finally, we demonstrate that MET coopts NMDAR2B to augment cell migration. This implies that MET harnesses the functionality of NMDAR2B to enhance the ability of cancer cells to migrate.

Calpain-mediated proteolysis as driver and modulator of polyglutamine toxicity.

Among posttranslational modifications, directed proteolytic processes have the strongest impact on protein integrity. They are executed by a variety of cellular machineries and lead to a wide range of molecular consequences. Compared to other forms of proteolytic enzymes, the class of calcium-activated calpains is considered as modulator proteases due to their limited proteolytic activity, which changes the structure and function of their target substrates. In the context of neurodegeneration and - in particular - polyglutamine disorders, proteolytic events have been linked to modulatory effects on the molecular pathogenesis by generating harmful breakdown products of disease proteins. These findings led to the formulation of the toxic fragment hypothesis, and calpains appeared to be one of the key players and auspicious therapeutic targets in Huntington disease and Machado Joseph disease. This review provides a current survey of the role of calpains in proteolytic processes found in polyglutamine disorders. Together with insights into general concepts behind toxic fragments and findings in polyglutamine disorders, this work aims to inspire researchers to broaden and deepen the knowledge in this field, which will help to evaluate calpain-mediated proteolysis as a unifying and therapeutically targetable posttranslational mechanism in neurodegeneration.

The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained genetically stable during the first 3 months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early-occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. IMPORTANCE The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions. The Alpha and Delta variants, which spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic compared to the original strain. Interestingly, Alpha and Delta both carried the mutations P681H/R in a cleavage site that made the spike more cleaved and more efficient at mediating viral fusion. We show here that variants with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of the D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite for the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma.

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

The emerging role of furin in neurodegenerative and neuropsychiatric diseases.

Furin is an important mammalian proprotein convertase that catalyzes the proteolytic maturation of a variety of prohormones and proproteins in the secretory pathway. In the brain, the substrates of furin include the proproteins of growth factors, receptors and enzymes. Emerging evidence, such as reduced FURIN mRNA expression in the brains of Alzheimer's disease patients or schizophrenia patients, has implicated a crucial role of furin in the pathophysiology of neurodegenerative and neuropsychiatric diseases. Currently, compared to cancer and infectious diseases, the aberrant expression of furin and its pharmaceutical potentials in neurological diseases remain poorly understood. In this article, we provide an overview on the physiological roles of furin and its substrates in the brain, summarize the deregulation of furin expression and its effects in neurodegenerative and neuropsychiatric disorders, and discuss the implications and current approaches that target furin for therapeutic interventions. This review may expedite future studies to clarify the molecular mechanisms of furin deregulation and involvement in the pathogenesis of neurodegenerative and neuropsychiatric diseases, and to develop new diagnosis and treatment strategies for these diseases.

Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death.

Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.