The NLRP3 Inflammasome Is a Major Cause of Acute Renal Failure Induced by Polypeptide Antibiotics.

Drug-induced acute renal failure (ARF) is a public health concern that hinders optimal drug therapy. However, pathological mechanisms of drug-induced ARF remain to be elucidated. Here, we show that a pathological process of drug-induced ARF is mediated by proinflammatory cross-talk between kidney tubular cells and macrophages. Both polymyxin B and colistin, polypeptide antibiotics, frequently cause ARF, stimulated the ERK and NF-κB pathways in kidney tubular cells, and thereby upregulated M-CSF and MCP-1, leading to infiltration of macrophages into the kidneys. Thereafter, the kidney-infiltrated macrophages were exposed to polypeptide antibiotics, which initiated activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Interestingly, blockade of the NLRP3 activation clearly ameliorated the pathology of ARF induced by polypeptide antibiotics, suggesting that a combination of the distinct cellular responses to polypeptide antibiotics in kidney tubular cells and macrophages plays a key role in the pathogenesis of colistin-induced ARF. Thus, our results provide a concrete example of how drugs initiate ARF, which may give insight into the underlying pathological process of drug-induced ARF.

LLPS of SQSTM1/p62 and NBR1 as outcomes of lysosomal stress response limits cancer cell metastasis.

Liquid droplet has emerged as a flexible intracellular compartment that modulates various cellular processes. Here, we uncover an antimetastatic mechanism governed by the liquid droplets formed through liquid-liquid phase separation (LLPS) of SQSTM1/p62 and neighbor of BRCA1 gene 1 (NBR1). Some of the tyrosine kinase inhibitors (TKIs) initiated lysosomal stress response that promotes the LLPS of p62 and NBR1, resulting in the spreading of p62/NBR1 liquid droplets. Interestingly, in the p62/NBR1 liquid droplet, degradation of RAS-related C3 botulinum toxin substrate 1 was accelerated by cellular inhibitor of apoptosis protein 1, which limits cancer cell motility. Moreover, the antimetastatic activity of the TKIs was completely overridden in p62/NBR1 double knockout cells both in vitro and in vivo. Thus, our results demonstrate a function of the p62/NBR1 liquid droplet as a critical determinant of cancer cell behavior, which may provide insight into both the clinical and biological significance of LLPS.

Membrane Dynamics and Cation Handling in Ferroptosis.

Ferroptosis, a regulated cell death hallmarked by excessive lipid peroxidation, is implicated in various (patho)physiological contexts. During ferroptosis, lipid peroxidation leads to a diverse change in membrane properties and the dysregulation of ion homeostasis via the cation channels, ultimately resulting in plasma membrane rupture. This review illuminates cellular membrane dynamics and cation handling in ferroptosis regulation.

The NLRP3 Inflammasome Works as a Sensor for Detecting Hypoactivity of the Mitochondrial Src Family Kinases.

Gefitinib (GF), the tyrosine kinase inhibitor (TKI) targeting epidermal growth factor receptor, initiates lung inflammation through the NLR family pyrin domain containing 3 (NLRP3) inflammasome. However, the molecular targets and mechanisms underlying the inflammatory action of GF remain unknown. In this study, we identified mitochondrial Src family kinases (mSFKs) as key determinants of GF-induced NLRP3 inflammasome activation. Comprehensive analysis of the TKIs revealed that all TKIs we tested act as potent agonists for the NLRP3 inflammasome in human monocytic THP-1 cells and bone marrow-derived macrophages. Moreover, these TKIs share a common off-target activity against the mSFKs, such as c-Src, Fgr, and Fyn. Interestingly, loss of each kinase spontaneously stimulated the NLRP3 inflammasome activation in THP-1 cells. These results together suggest that NLRP3 senses hypoactivity of the mSFKs that is responsible for mitochondrial dysfunction. Thus, our findings demonstrate a mechanistic link between the NLRP3 inflammasome and mSFKs, which, to our knowledge, provides insights into a novel molecular basis and cellular function of the NLRP3 inflammasome.

Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos.

Reactive sulfur species (RSS) have emerged as key regulators of protein quality control. However, the mechanisms by which RSS contribute to cellular processes are not fully understood. In this study, we identified a novel function of RSS in preventing parthanatos, a nonapoptotic form of cell death that is induced by poly (ADP-ribose) polymerase-1 and mediated by the aggresome-like induced structures (ALIS) composed of SQSTM1/p62. We found that sodium tetrasulfide (Na2S4), a donor of RSS, strongly suppressed oxidative stress-dependent ALIS formation and subsequent parthanatos. On the other hand, the inhibitors of the RSS-producing enzymes, such as 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase, clearly enhanced ALIS formation and parthanatos. Interestingly, we found that Na2S4 activated heat shock factor 1 by promoting its dissociation from heat shock protein 90, leading to accelerated transcription of HSP70. Considering that the genetic deletion of HSP70 allowed the enhanced ALIS formation, these findings suggest that RSS prevent parthanatos by specifically suppressing ALIS formation through induction of HSP70. Taken together, our results demonstrate a novel mechanism by which RSS prevent cell death, as well as a novel physiological role of RSS in contributing to protein quality control through HSP70 induction, which may lead to better understanding of the bioactivity of RSS.

The Distinct Roles of LKB1 and AMPK in p53-Dependent Apoptosis Induced by Cisplatin.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase that acts as a key tumor suppressor protein by activating its downstream kinases, such as AMP-activated protein kinase (AMPK). However, the regulatory actions of LKB1 and AMPK on DNA damage response (DDR) remain to be explored. In this study, we investigated the function of LKB1 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that LKB1 stabilizes and activates p53 through the c-Jun N-terminal kinase (JNK) pathway, which promotes cisplatin-induced apoptosis in human fibrosarcoma cell line HT1080. On the other hand, we found that AMPKα1 and α2 double knockout (DKO) cells showed enhanced stabilization of p53 and increased susceptibility to apoptosis induced by cisplatin, suggesting that AMPK negatively regulates cisplatin-induced apoptosis. Moreover, the additional stabilization of p53 and subsequent apoptosis in AMPK DKO cells were clearly canceled by the treatment with the antioxidants, raising the possibility that AMPK suppresses the p53 activation mediated by oxidative stress. Thus, our findings unexpectedly demonstrate the reciprocal regulation of p53 by LKB1 and AMPK in DDR, which provides insights into the molecular mechanisms of DDR.

trans-Fatty Acids as an Enhancer of Inflammation and Cell Death: Molecular Basis for Their Pathological Actions.

trans-Fatty acids (TFAs) are food-derived fatty acids that possess one or more trans double bonds between carbon atoms. Compelling epidemiological and clinical evidence has demonstrated the association of TFA consumption with various diseases, such as cardiovascular diseases, and neurodegenerative diseases. However, the underlying etiology is poorly understood since the mechanisms of action of TFAs remain to be clarified. Previous studies have shown that single treatment with TFAs induce inflammation and cell death, but to a much lesser extent than saturated fatty acids (SFAs) that are well established as a risk factor for diseases linked with inflammation and cell death, which cannot explain the particularly higher association of TFAs with atherosclerosis than SFAs. In our series of studies, we have established the role of TFAs as an enhancer of inflammation and cell death. We found that pretreatment with TFAs strongly promoted apoptosis induced by either extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from damaged cells, or DNA damaging-agents, including doxorubicin and cisplatin, thorough enhancing activation of the stress-responsive mitogen-activated protein (MAP) kinase p38/c-jun N-terminal kinase (JNK) pathways; pretreatment with SFAs or cis isomers of TFAs had only minor or no effect, suggesting the uniqueness of the pro-apoptotic role of TFAs among fatty acids. Our findings will provide an insight into understanding of the pathogenesis mechanisms, and open up a new avenue for developing prevention strategies and therapies for TFA-related diseases.

The E3 Ubiquitin-Protein Ligase RNF4 Promotes TNF-α-Induced Cell Death Triggered by RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) is a key component of the tumor necrosis factor (TNF) receptor signaling complex that regulates both pro- and anti-apoptotic signaling. The reciprocal functions of RIPK1 in TNF signaling are determined by the state of the posttranslational modifications (PTMs) of RIPK1. However, the underlying mechanisms associated with the PTMs of RIPK1 are unclear. In this study, we found that RING finger protein 4 (RNF4), a RING finger E3 ubiquitin ligase, is required for the RIPK1 autophosphorylation and subsequent cell death. It has been reported that RNF4 negatively regulates TNF-α-induced activation of the nuclear factor-κB (NF-κB) through downregulation of transforming growth factor ß-activated kinase 1 (TAK1) activity, indicating the possibility that RNF4-mediated TAK1 suppression results in enhanced sensitivity to cell death. However, interestingly, RNF4 was needed to induce RIPK1-mediated cell death even in the absence of TAK1, suggesting that RNF4 can promote RIPK1-mediated cell death without suppressing the TAK1 activity. Thus, these observations reveal the existence of a novel mechanism whereby RNF4 promotes the autophosphorylation of RIPK1, which provides a novel insight into the molecular basis for the PTMs of RIPK1.

Elaidic Acid Potentiates Extracellular ATP-Induced Apoptosis via the P2X7-ROS-ASK1-p38 Axis in Microglial Cell Lines.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X7), or Ca2+/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

TAK1 Mediates ROS Generation Triggered by the Specific Cephalosporins through Noncanonical Mechanisms.

It is known that a wide variety of antibacterial agents stimulate generation of reactive oxygen species (ROS) in mammalian cells. However, its mechanisms are largely unknown. In this study, we unexpectedly found that transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) is involved in the generation of mitochondrial ROS (mtROS) initiated by cefotaxime (CTX), one of specific antibacterial cephalosporins that can trigger oxidative stress-induced cell death. TAK1-deficient macrophages were found to be sensitive to oxidative stress-induced cell death stimulated by H2O2. Curiously, however, TAK1-deficient macrophages exhibited strong resistance to oxidative stress-induced cell death stimulated by CTX. Microscopic analysis revealed that CTX-induced ROS generation was overridden by knockout or inhibition of TAK1, suggesting that the kinase activity of TAK1 is required for CTX-induced ROS generation. Interestingly, pharmacological blockade of the TAK1 downstream pathways, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, did not affect the CTX-induced ROS generation. In addition, we observed that CTX promotes translocation of TAK1 to mitochondria. Together, these observations suggest that mitochondrial TAK1 mediates the CTX-induced mtROS generation through noncanonical mechanisms. Thus, our data demonstrate a novel and atypical function of TAK1 that mediates mtROS generation triggered by the specific cephalosporins.

Validation of prognostic impact of number of extrathoracic metastases according to the eighth TNM classification: a single-institution retrospective study in Japan.

BACKGROUND: In the eighth edition of the TNM classification of lung cancer, the M1b and M1c descriptors are newly defined by the number of extrathoracic metastases. To verify the prognostic value of these descriptors in Japan, we reclassified our cases and re-evaluated prognosis in M1b and M1c patients. METHODS: All non-small cell lung cancer (NSCLC) patients with extrathoracic metastases who visited Saitama Medical Center from 2010 to 2016 were evaluated, divided according to the eighth edition of the TNM classification criteria into two groups (M1b, patients with single extrathoracic metastasis, and M1c, patients with multiple extrathoracic metastases), and followed up until December 31, 2017. Survival time analysis was performed using the Kaplan-Meier method, and between-group differences in overall survival time (OS) were evaluated by the log-rank test. RESULTS: A total of 231 NSCLC patients were divided into 57 patients with M1b and 174 with M1c. Median OS was 15.2 months (95% confidence interval [CI]: 9.3-19.9) and 7.3 months (95% CI 5.7-10.7) for M1b and M1c, respectively, with no significant between-group difference (P = 0.239). However, after excluding patients with epidermal growth factor receptor (EGFR) mutation or echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene, median OS was 12.9 months (95% CI 7.2-19.9) for M1b and 5.4 months (95% CI 3.8-6.3) for M1c, respectively, showing a significant difference (P = 0.029). CONCLUSIONS: The effect of therapy directed toward EGFR mutation or EML4-ALK fusion gene might obscure the significant prognostic difference between M1b and M1c.

Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans.

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK.

trans-Fatty acids promote proinflammatory signaling and cell death by stimulating the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway.

Food-borne trans-fatty acids (TFAs) are mainly produced as byproducts during food manufacture. Recent epidemiological studies have revealed that TFA consumption is a major risk factor for various disorders, including atherosclerosis. However, the underlying mechanisms in this disease etiology are largely unknown. Here we have shown that TFAs potentiate activation of apoptosis signal-regulating kinase 1 (ASK1) induced by extracellular ATP, a damage-associated molecular pattern leaked from injured cells. Major food-associated TFAs such as elaidic acid (EA), linoelaidic acid, and trans-vaccenic acid, but not their corresponding cis isomers, dramatically enhanced extracellular ATP-induced apoptosis, accompanied by elevated activation of the ASK1-p38 pathway in a macrophage-like cell line, RAW264.7. Moreover, knocking out the ASK1-encoding gene abolished EA-mediated enhancement of apoptosis. We have reported previously that extracellular ATP induces apoptosis through the ASK1-p38 pathway activated by reactive oxygen species generated downstream of the P2X purinoceptor 7 (P2X7). However, here we show that EA did not increase ATP-induced reactive oxygen species generation but, rather, augmented the effects of calcium/calmodulin-dependent kinase II-dependent ASK1 activation. These results demonstrate that TFAs promote extracellular ATP-induced apoptosis by targeting ASK1 and indicate novel TFA-associated pathways leading to inflammatory signal transduction and cell death that underlie the pathogenesis and progression of TFA-induced atherosclerosis. Our study thus provides insight into the pathogenic mechanisms of and proposes potential therapeutic targets for these TFA-related disorders.

Heterodimerization of Group†I Ribozymes Enabling Exon Recombination through Pairs of Cooperative trans-Splicing Reactions.

Group I (GI) self-splicing ribozymes are attractive tools for biotechnology and synthetic biology. Several trans-splicing and related reactions based on GI ribozymes have been developed for the purpose of recombining their target mRNA sequences. By combining trans-splicing systems with rational modular engineering of GI ribozymes it was possible to achieve more complex editing of target RNA sequences. In this study we have developed a cooperative trans-splicing system through rational modular engineering with use of dimeric GI ribozymes derived from the Tetrahymena group I intron ribozyme. The resulting pairs of ribozymes exhibited catalytic activity depending on their selective dimerization. Rational modular redesign as performed in this study would facilitate the development of sophisticated regulation of double or multiple trans-splicing reactions in a cooperative manner.

Post-Translational Modifications of the TAK1-TAB Complex.

Transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that is activated by growth factors and cytokines such as TGF-ß, IL-1ß, and TNF-α, and mediates a wide range of biological processes through activation of the nuclear factor-κB (NF-κB) and the mitogen-activated protein (MAP) kinase signaling pathways. It is well established that activation status of TAK1 is tightly regulated by forming a complex with its binding partners, TAK1-binding proteins (TAB1, TAB2, and TAB3). Interestingly, recent evidence indicates the importance of post-translational modifications (PTMs) of TAK1 and TABs in the regulation of TAK1 activation. To date, a number of PTMs of TAK1 and TABs have been revealed, and these PTMs appear to fine-tune and coordinate TAK1 activities depending on the cellular context. This review therefore focuses on recent advances in the understanding of the PTMs of the TAK1-TAB complex.

Multidisciplinary treatment for prepubertal juvenile myasthenia gravis with crisis.

The management of juvenile myasthenia gravis (MG) remains controversial. We report herein the case of a 12-year-old girl with prepubertal juvenile MG with respiratory crisis who underwent thymectomy following methylprednisolone pulse therapy. The patient initially developed progressively worsening fatigability, eyelid ptosis, and diplopia, followed by worsening generalized weakness, dysphagia, and dyspnea. Even after i.v. immunoglobulin, the patient presented with rapid onset of severe dyspnea requiring respiratory support with mechanical ventilation and was graded as Myasthenia Gravis Foundation of America class V. After a course of i.v. methylprednisolone pulse therapy, successful control of respiratory crisis was achieved, and trans-sternal thymectomy was performed. Partial remission was achieved postoperatively with oral pyridostigmine without immunosuppressive agents such as steroids or calcineurin inhibitors for 18 months after thymectomy. Early thymectomy following induction methylprednisolone pulse therapy might be a treatment option for prepubertal juvenile MG with severe respiratory crisis.

Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor 1 (BIG1) Governs the Recruitment of Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) to Tumor Necrosis Factor Receptor 1 (TNFR1) Signaling Complexes.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.

Mg2+-dependent interactions of ATP with the cystathionine-ß-synthase (CBS) domains of a magnesium transporter.

Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg(2+) transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine-ß-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg(2+) efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg(2+) efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg(2+), which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg(2+) for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg(2+) efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg(2+) in cells, may account for the rapid Mg(2+) transport by CNNM family proteins.

Basolateral sorting of the Mg²âº transporter CNNM4 requires interaction with AP-1A and AP-1B.

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of µ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the µ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with µ1A and µ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.

Identification of small subunit of serine palmitoyltransferase a as a lysophosphatidylinositol acyltransferase 1-interacting protein.

Lysophosphatidylinositol acyltransferase 1 (LPIAT1), also known as MBOAT7, is a phospholipid acyltransferase that selectively incorporates arachidonic acid (AA) into the sn-2 position of phosphatidylinositol (PI). We previously demonstrated that LPIAT1 regulates AA content in PI and plays a crucial role in brain development in mice. However, how LPIAT1 is regulated and which proteins function cooperatively with LPIAT1 are unknown. In this study, using a split-ubiquitin membrane yeast two-hybrid system, we identified the small subunit of serine palmitoyltransferase a (ssSPTa) as an LPIAT1-interacting protein. ssSPTa co-immunoprecipitated and colocalized with LPIAT1 in cultured mammalian cells. Knockdown of ssSPTa decreased the LPIAT1-dependent incorporation of exogenous AA into PI but did not affect the in vitro enzyme activity of LPIAT1 in the microsomal fraction. Interestingly, knockdown of ssSPTa decreased the protein level of LPIAT1 in the crude mitochondrial fraction but not in total homogenate or the microsomal fraction. LPIAT1 was localized to the mitochondria-associated membrane (MAM), where AA-selective acyl-CoA synthetase is enriched. These results suggest that ssSPTa plays a role in fatty acid remodeling of PI, probably by facilitating the MAM localization of LPIAT1.