Michal Lichtenstein

The mitochondrial phosphate carrier (mPiC), encoded by the nuclear gene SLC25A3, is synthesized with an N-terminus mitochondrial targeting sequence (MTS), enabling its import into the mitochondria. mPiC imports inorganic phosphate (P_i) into the mitochondrial matrix for ATP production and other matrix phosphorylation reactions, as well as regulates mitochondrial Ca²⁺ uptake and buffering of matrix Ca²⁺. PiC also imports copper (Cu), crucial to COX subunit holoenzyme assembly. Variants in SLC25A3 exist and lead to mPiC deficiency (MPCD), cause a rare autosomal recessive disease with no current cure; patients with MPCD usually die within the first year of life. We have developed a novel therapeutic approach using TAT-mPiC fusion protein for cellular delivery since the TAT peptide enables delivery of proteins across biological membranes. We designed, produced, and purified the TAT-mPiC fusion protein. The fusion protein is delivered into the mitochondria and localizes within the mIM, its natural cellular location, as a processed protein. Treatment of mPiC-knockdown cells with TAT-mPiC fusion protein increased cell growth and improved bioenergetic capabilities, as measured by oxygen consumption rate (OCR), ATP production, and reduction in lactate secretion. Most importantly, TAT-mPiC restored P_i and Cu delivery into the mitochondrial matrix. TAT-mPiC fusion protein also restored the mitochondrial activity of cells harboring various mitochondrial defects. This study presents the first successful delivery of a mitochondrial transmembrane carrier using the TAT-fusion system, offering a potential early treatment strategy for newborns with mPiC deficiency.

Background: Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein. Methods and Results: In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA–degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model. Conclusions: Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway.

Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.

Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such eclipsed distribution: one systematic and unbiased and the other based on Mitochondrial Targeting Signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein-family of the Triose-phosphate DeHydrogenases (TDH), and prove that indeed their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.

We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer’s disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy.

To overcome the lack of specificity of cancer therapeutics and thus create a more potent and effective treatment, we developed a novel chimeric protein, IL2-Smurf2. Here, we describe the production of this chimeric IL2-Smurf2 protein and its variants, with inactive or over-active killing components. Using Western blots, we demonstrated the chimeric protein’s ability to specifically enter target cells alone. After entering the cells, the protein showed biological activity, causing cell death that was not seen with an inactive variant, and that was shown to be apoptotic. The chimeric protein also proved to be active as an E3 ligase, as demonstrated by testing total ubiquitination levels along with targeted ubiquitination for degradation. Finally, we tested IL2-Smurf2 and its variants in an in vivo mouse model of leukemia and demonstrated its potential as a drug for the targeted treatment of cancer cells. In the course of this work, we established for the first time the feasibility of the use of Smurf2 as a killing component in chimeric targeting proteins. Utilizing the IL2 cytokine to target cells overexpressing IL-2R and Smurf2 to cause protein degradation, we were able to produce a chimeric protein with dual functionality which causes targeted cell death.

Fumarate hydratase (FH) is an evolutionary conserved TCA cycle enzyme that reversibly catalyzes the hydration of fumarate to L-malate and has a moonlight function in the DNA damage response (DDR). Interestingly, FH has a contradictory cellular function, as it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis. Here, we found that in both non-cancerous (HEK-293T) and cancerous cell lines (HepG2), the cell response to FH loss is separated into two distinct time frames based on cell proliferation and DNA damage repair. During the early stages of FH loss, cell proliferation rate and DNA damage repair are inhibited. However, over time the cells overcome the FH loss and form knockout clones, indistinguishable from WT cells with respect to their proliferation rate. Due to the FH loss effect on DNA damage repair, we assumed that the recovered cells bear adaptive mutations. Therefore, we applied whole-exome sequencing to identify such mutated genes systematically. Indeed, we identified recurring mutations in genes belonging to central oncogenic signaling pathways, such as JAK/STAT3, which we validated in impaired FH-KO clones. Intriguingly, we demonstrate that these adaptive mutations are responsible for FH-KO cell proliferation under TCA cycle malfunction.

Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering enzymes/proteins into cells. In this review, we describe the potential therapeutic usages of CPPs (mainly trans-activator of transcription protein, TAT) in enabling the uptake of biologically active proteins/enzymes needed in cases of protein/enzyme deficiency, concentrating on mitochondrial diseases and on the import of enzymes or peptides in order to destroy pathogenic cells, focusing on cancer cells.

Cancer-dependent metabolic rewiring is often manifested by selective expression of enzymes essential for the transformed cells’ viability. However, the metabolic variations between normal and transformed cells are not fully characterized, and therefore, a systematic analysis will result in the identification of unknown cellular mechanisms crucial for tumorigenesis. Here, we applied differential gene expression transcriptome analysis to examine the changes in metabolic gene profiles between a wide range of normal tissues and cancer samples. We found that, in contrast to normal tissues which exhibit a tissue-specific expression profile, cancer samples are more homogenous despite their diverse origins. This similarity is due to a “proliferation metabolic signature” (PMS), composed of 158 genes (87 upregulated and 71 downregulated gene sets), where 143 are common to all proliferative cells but 15 are cancer specific. Intriguingly, the PMS gene set is enriched for genes encoding rate-limiting enzymes, and its upregulated set with genes associated with poor patient outcome and essential genes. Among these essential genes is ribulose-5-phosphate-3-epimerase (RPE), which encodes a pentose phosphate pathway enzyme and whose role in cancer is still unclear. Collectively, we identified a set of metabolic genes that can serve as novel cancer biomarkers and potential targets for drug development.

Metabolic reprogramming as a downstream result of oncogenic signaling pathways has been described as a hallmark of cancer. Here, we describe a reverse scenario in which a metabolic enzyme regulates cancer cell behavior by triggering a signaling pathway. We find that glutathione peroxidase 8 (GPX8), a poorly characterized redox enzyme that resides in the endoplasmic reticulum, is upregulated during the epithelial-to-mesenchymal (EMT) program in HMLE and A549 cells. In cancer patients, high tumor levels of GPX8 correlate with mesenchymal markers and poor patient outcome. Strikingly, GPX8 knockout in mesenchymal-like cells results in an epithelial-like morphology, downregulation of EMT characteristics, loss of cancer stemness features, and impeded tumor initiation in mice. We determine the mechanism governing this reduction in cancer aggressiveness is through the repression of crucial autocrine factors, in particular, interleukin-6 (IL-6). Specifically, GPX8 knockout impairs IL-6-driven activation of the JAK-STAT3 signaling pathway, a critical regulator of a cancer-aggressive state. Altogether, we uncover the GPX8-IL-6 axis as a novel metabolic-inflammatory pathway that acts as a robust EMT activator and program to induce aggressive cancer cell characteristics.