Tetramerization Domain Research Articles

Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain

During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37°C to 15°C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass.

Molecular cloning, characterization and expression analysis of p53 from turbot Scophthalmus maximus and its response to thermal stress.

The tumor suppressor protein, p53 plays a crucial role in protecting genetic integrity. Once activated by diverse cell stresses, p53 reversibly activates downstream target genes to regulate cell cycle and apoptosis. However, few studies have investigated the effects of thermal stress in turbot, specifically the p53 signaling pathway. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot p53 gene (Sm-p53) and perform bioinformatics analysis. The results showed that the cDNA of the Sm-p53 gene was 2928 bp in length, encoded a 381 amino acid protein, with a theoretical isoelectric point of 6.73. It was composed of a DNA binding and a tetramerization domain. Expression of Sm-p53 in different tissues was detected and quantified by qRT-PCR, and was highest in the liver. We also investigated the expression profiles of Sm-p53 in different tissue and TK cells after thermal stress. These result suggested that Sm-p53 plays a key role, and provides a theoretical basis for Sm-p53 changes in environmental stress responses in the turbot.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

An Ancient Mutation in the TPH1 Gene is Consistent with the Changes in Mammalian Reproductive Rhythm.

The reproductive rhythm undergoes several changes during the evolution of mammals to adapt to local environmental changes. Although the critical roles of melatonin (MLT) in the formation of reproductive rhythm have been well established, the genetic basis for the changes of reproductive rhythm remains uncertain. Here, we constructed the phylogenetic trees of 13 melatonin synthesis, metabolism and receptor genes, estimated their divergence times, and calculated their selection pressures. Then, we evaluated the effect of positively selected and functionally related mutations on protein activity. Our results showed that there were significant positive selection sites in the three major genes, including tryptophan hydroxylase 1 (TPH1), tryptophan hydroxylase 2 (TPH2) and indoleamine-2,3-dioxygenase 1 (IDO1) that are involved in melatonin synthesis, metabolism and function. At the protein level, amino acids at the 442nd site of TPH1 protein and the 194th, 286th, 315th and 404th sites of IDO1 protein were under positive selection, and the variants of the amino acid in these sites might lead to the changes in protein function. Remarkably, the 442nd site of these positive selection sites is in the tetramerization domain of TPH1 protein, and it is proline or leucine. At this site, 89.5% of the amino acid of non-seasonal reproducing mammals was proline, while that of 88.9% of seasonal reproducing mammals was leucine. This variation of the amino acid was derived from the T/C polymorphism at the 1325th site of the TPH1 gene coding sequence, which significantly altered the TPH1 activity (p < 0.01). Interestingly, the predicted age of the allele C in the mammalian genome appeared about 126.6 million years ago, and allele T appeared about 212.6 million years ago, indicating that the evolution of the TPH1 gene was affected by the two mammalian split events and the K-T extinction event. In conclusion, the T/C polymorphism at the 1325th site in the TPH1 gene coding sequence altered TPH1 activity, suggesting that this polymorphism is consistent with the reproductive rhythm of mammals.

Phenoxyethyl Piperidine/Morpholine Derivatives as PAS and CAS Inhibitors of Cholinesterases: Insights for Future Drug Design

Acetylcholinesterase (AChE) catalyzes the conversion of Aβ peptide to its aggregated form and the peripheral anionic site (PAS) of AChE is mainly involved in this phenomenon. Also catalytic active site (CAS) of donepezil stimulates the break-down of acetylcholine (ACh) and depletion of ACh in cholinergic synapses are well established in brains of patients with AD. In this study, a set of compounds bearing phenoxyethyl amines were synthesized and their inhibitory activity toward electric eel AChE (eeAChE) and equine butyrylcholinesterase (eqBuChE) were evaluated. Molecular dynamics (MD) was employed to record the binding interactions of best compounds against human cholinesterases (hAChE and hBuChE) as well as donepezil as reference drug. In vitro results revealed that compound 5c is capable of inhibiting eeAChE activity at IC50 of 0.50 µM while no inhibitory activity was found for eqBuChE for up to 100 µM concentrations. Compound 5c, also due to its facile synthesis, small structure and high selectivity for eeAChE would be very interesting candidate in forthcoming studies. The main interacting parts of compound 5c and compound 7c (most potent eeAChE and eqBuChE inhibitors respectively) with receptors which confer selectivity for AChE and BuChE inhibition were identified, discussed, and compared with donepezil’s interactions. Also during MD simulation it was discovered for the first time that binding of substrates like donepezil to dual CAS and PAS or solely CAS region might have a suppressive impact on 4-α-helical bundles near the tryptophan amphiphilic tetramerization (WAT) domain of AChE and residues which are far away from AChE active site. The results proposed that residues involved in donepezil interactions (Trp86 and Phe295) which are located in CAS and mid-gorge are the mediator of conformational changes in whole protein structure.

KCTD15 is overexpressed in human childhood B-cell acute lymphoid leukemia

Leukemic cells originate from the malignant transformation of undifferentiated myeloid/lymphoid hematopoietic progenitors normally residing in bone marrow. As the precise molecular mechanisms underlying this heterogeneous disease are yet to be disclosed, the identification and the validation of novel actors in leukemia is of extreme importance. Here, we show that KCTD15, a member of the emerging class of KCTD ((K)potassium Channel Tetramerization Domain containing) proteins, is strongly upregulated in patients affected by B-cell type acute lymphoblastic leukemia (B-ALL) and in continuous cell lines (RS4;11, REH, TOM-1, SEM) derived from this form of childhood leukemia. Interestingly, KCTD15 downregulation induces apoptosis and cell death suggesting that it has a role in cellular homeostasis and proliferation. In addition, stimulation of normal lymphocytes with the pokeweed mitogen leads to increased KCTD15 levels in a fashion comparable to those observed in proliferating leukemic cells. In this way, the role of KCTD15 is likely not confined to the B-ALL pathological state and extends to activation and proliferation of normal lymphocytes. Collectively, data here presented indicate that KCTD15 is an important and hitherto unidentified player in childhood lymphoid leukemia, and its study could open a new scenario for the identification of altered and still unknown molecular pathways in leukemia.

Identification of new genes of pleomorphic adenoma.

Pleomorphic adenoma is the most common salivary gland neoplasm with a variety of histologic appearances. Due to this diversity, precise preoperative diagnosis through fine needle aspiration cytology is difficult.This study sought to identify the differentially expressed genes in pleomorphic adenoma to aid precise diagnosis and clarify the mechanism of tumorigenesis.Suppressive subtractive hybridization was performed on pleomorphic adenoma tissues and the corresponding normal salivary gland tissues to screen of the differential expression of genes in pleomorphic adenoma.Four known genes (microfibrillar associated protein 4 [MFAP4], dystonin [DST], solute carrier family 35 [SLC35], and potassium channel tetramerization domain containing 15 [KCTD15]) were differentially expressed in the tumors compared with the genes in normal tissues. The expression profiles were further confirmed in 15 pleomorphic adenoma and corresponding normal salivary gland tissues by quantitative real-time reverse transcription-polymerase chain reaction.MFAP4, DST, SLC35, and KCTD15 gene expression could be potential biomarkers of pleomorphic adenoma for precise diagnosis.

Landscape of TP53 Mutations in MPN

Landscape of TP53 Mutations in MPN

Structure of the human metapneumovirus polymerase phosphoprotein complex.

Summary paragraphRespiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elder adults1. Neither a vaccine nor an effective antiviral therapy exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the nucleoprotein-RNA (N-RNA) template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase (PRNTase) and cap-specific methyltransferases (MTases) activities2,3. How P interacts with L and mediates association with the free form of N and with the ribonucleoprotein (RNP) is not clear for HMPV or other prominent human pathogens including measles, Ebola and rabies viruses. Here, we report a cryo-EM reconstruction showing the ring-shaped structure of the polymerase and capping domains of HMPV L, bound with a tetramer of P. The connector and MTase domains are mobile with respect to the core. The putative priming loop important for initiation of RNA synthesis is fully retracted, leaving space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å2 of molecular surface area in the interface. Two of the four helices forming the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein like tentacles. The structural versatility of the four P protomers, which are largely disordered in their free state, demonstrates an example of a “folding-upon-partner-binding” mechanism for carrying-out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and should accelerate the design of specific antiviral drugs.

The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein

The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.

KCTD15 inhibits the Hedgehog pathway in Medulloblastoma cells by increasing protein levels of the oncosuppressor KCASH2

Medulloblastoma (MB) is the most common malignant childhood brain tumor. About 30% of all MBs belong to the I molecular subgroup, characterized by constitutive activation of the Sonic Hedgehog (Hh) pathway. The Hh pathway is involved in several fundamental processes during embryogenesis and in adult life and its deregulation may lead to cerebellar tumorigenesis. Indeed, Hh activity must be maintained via a complex network of activating and repressor signals. One of these repressor signals is KCASH2, belonging to the KCASH family of protein, which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation. KCASH2 leads HDAC1 to degradation, allowing hyperacetylation and inhibition of transcriptional activity of Gli1, the main effector of the Hh pathway. In turn, the KCASH2 loss leads to persistent Hh activity and eventually tumorigenesis. In order to better characterize the physiologic role and modulation mechanisms of KCASH2, we have searched through a proteomic approach for new KCASH2 interactors, identifying Potassium Channel Tetramerization Domain Containing 15 (KCTD15). KCTD15 is able to directly interact with KCASH2, through its BTB/POZ domain. This interaction leads to increase KCASH2 stability which implies a reduction of the Hh pathway activity and a reduction of Hh-dependent MB cells proliferation. Here we report the identification of KCTD15 as a novel player in the complex network of regulatory proteins, which modulate Hh pathway, this could be a promising new target for therapeutic approach against MB.

Computational identification of key genes that may regulate gene expression reprogramming in Alzheimer’s patients

The dementia epidemic is likely to expand worldwide as the aging population continues to grow. A better understanding of the molecular mechanisms that lead to dementia is expected to reveal potentially modifiable risk factors that could contribute to the development of prevention strategies. Alzheimer’s disease is the most prevalent form of dementia. Currently we only partially understand some of the pathophysiological mechanisms that lead to development of the disease in aging individuals. In this study, Switch Miner software was used to identify key switch genes in the brain whose expression may lead to the development of Alzheimer’s disease. The results indicate that switch genes are enriched in pathways involved in the proteasome, oxidative phosphorylation, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and metabolism in the hippocampus and posterior cingulate cortex. Network analysis identified the krupel like factor 9 (KLF9), potassium channel tetramerization domain 2 (KCTD2), Sp1 transcription factor (SP1) and chromodomain helicase DNA binding protein 1 (CHD1) as key transcriptional regulators of switch genes in the brain of AD patients. These transcriptions factors have been implicated in conditions associated with Alzheimer’s disease, including diabetes, glucocorticoid signaling, stroke, and sleep disorders. The specific pathways affected reveal potential modifiable risk factors by lifestyle changes.

Targeting the γ-Aminobutyric Acid Type B (GABAB) Receptor Complex: Development of Inhibitors Targeting the K+ Channel Tetramerization Domain (KCTD) Containing Proteins/GABAB Receptor Protein–Protein Interaction

Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein-protein interaction of the GABAB receptor complex-the interaction with KCTD proteins. By employing the μSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.

No Significant Association between Radiation Therapy and Subsequent Malignancies in Patients with Li-Fraumeni Syndrome: A Multi-Institutional Hereditary Cancer Registry Study

Li-Fraumeni Syndrome (LFS) is a rare cancer-predisposing disease caused by germline mutations in a potent tumor suppressor gene calledTP53. Accordingly, LFS patients carry a high lifetime risk of developing multiple primary cancers. Conventional wisdom and prior work have suggested that the risk of developing a subsequent malignancy is increased by the use of radiation therapy (RT) during primary or secondary cancer treatment. This risk, however, is not well characterized. Here we describe the risk of subsequent malignancy and cancer-related death in LFS patients after undergoing RT for a first or second primary cancer. We reviewed a cohort of patients in a multi-institution hereditary cancer registry with germline TP53 mutations. We assessed the rate of subsequent malignancy and death in patients who received RT (RT group) as part of their cancer treatment against those who did not (non-RT group). We further evaluated outcomes after RT with respect to p53 mutation type. Among 41 LFS patients (14 males and 28 females from 28 different families), 22 TP53mutations were present. 61% (25) and 7% (3) of patients had missense mutations in the DNA binding domain and tetramerization domain of p53, respectively, and 27% (11) had truncating mutations caused by nonsense (3), frameshift (1), splice site (2), or deletion (5) mutations. The median age of first cancer diagnosis was 22 years. 13 patients received RT with curative intent as part of their cancer treatment which included 3 for locally recurrent disease. The median time to follow up after RT was 5.0 years. 61% of patients in the RT group compared to 40% of patients in the non-RT group developed a subsequent malignancy (P=0.31). The median time to subsequent malignancy in the RT group and non-RT group was 3.2 years and 4.3 years respectively. Although 4 patients developed a subsequent malignancy within the radiation field, all were of the same initial histology and none of the cancers had features of an RT-induced secondary malignancy. To date, 8 deaths have occurred in the RT group (median age 36.5 years) and 2 deaths in the non-RT group (median age 30.5 years). The 5-year overall survival (OS) for the RT group was 54% compared to 95% in the non-RT group (P=0.033). Notably, there was no association between the type of p53 mutation and the rate of subsequent malignancy after RT. LFS patients who received RT for a primary cancer suffered from subsequent malignancies and death at a higher rate than those who did not receive RT. However, these results must be interpreted with caution as the majority of patients within the RT-treated group had more aggressive cancers with a higher risk of recurrence. We found that all 4 in-field malignancies in the RT-group likely represent locally recurrent disease and not RT-induced malignancies. Accordingly, the higher rate of mortality in the RT-group is likely a reflection of more aggressive cancer diagnoses and not their treatment.

KCTD1: A novel modulator of adipogenesis through the interaction with the transcription factor AP2α

Adipogenesis has an important role in regulating energy balance, tissue homeostasis and disease pathogenesis. 3T3-L1 preadipocytes have been widely used as an in vitro model for studying adipocyte differentiation. We here show that KCTD1, a member of the potassium channel containing tetramerization domain proteins, plays an active role in adipogenesis. In particular, we show KCTD1 expression 3T3-L1 cells increases upon adipogenesis induction. Treatment of 3T3-L1 preadipocytes with Kctd1-specific siRNA inhibited the differentiation, as indicated by reduction of expression of the specific adipogenic markers C/ebpα, Pparγ2, Glut4, and Adiponectin. Moreover, we also show that the protein physically interacts with the transcription factor AP2α, a known inhibitor of adipogenesis, both in vitro and in cells. Interestingly, our data indicate that KCTD1 promotes adipogenesis through the interaction with AP2α and by removing it from the nucleus. Collectively, these findings disclose a novel role for KCTD1 and pave the way for novel strategies aimed at modulating adipogenesis.

Bipartite interface of the measles virus phosphoprotein X domain with the large polymerase protein regulates viral polymerase dynamics.

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility.

The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABAB Receptor.

Several recent investigations have demonstrated that members of the KCTD (Potassium Channel Tetramerization Domain) protein family are involved in fundamental processes. However, the paucity of structural data available on these proteins has frequently prevented the definition of their biochemical role(s). Fortunately, this scenario is rapidly changing as, in very recent years, several crystallographic structures have been reported. Although these investigations have provided very important insights into the function of KCTDs, they have also raised some puzzling issues. One is related to the observation that the BTB (broad-complex, tramtrack, and bric-à-brac) domain of these proteins presents a remarkable structural versatility, being able to adopt a variety of oligomeric states. To gain insights into this intriguing aspect, we performed extensive molecular dynamics simulations on several BTB domains of KCTD proteins in different oligomeric states (monomers, dimers, tetramers, and open/close pentamers). These studies indicate that KCTD-BTB domains are stable in the simulation timescales, even in their monomeric forms. Moreover, simulations also show that the dynamic behavior of open pentameric states is strictly related to their functional roles and that different KCTDs may form stable hetero-oligomers. Molecular dynamics (MD) simulations also provided a dynamic view of the complex formed by KCTD16 and the GABAB2 receptor, whose structure has been recently reported. Finally, simulations carried out on the isolated fragment of the GABAB2 receptor that binds KCTD16 indicate that it is able to assume the local conformation required for the binding to KCTD.

P32 is a negative regulator of p53 tetramerization and transactivation.

p53 is a sequence‐specific transcription factor, and proper regulation of p53 transcriptional activity is critical for orchestrating different tumor‐suppressive mechanisms. p32 is a multifunctional protein which interacts with a large number of viral proteins and transcription factors. Here, we investigate the effect of p32 on p53 transactivation and identify a novel mechanism by which p32 alters the functional characteristics of p53. Specifically, p32 attenuates p53‐dependent transcription through impairment of p53 binding to its response elements on target genes. Upon p32 expression, p53 levels bound at target genes are decreased, and p53 target genes are inactivated, strongly indicating that p32 restricts p53 occupancy and function at target genes. The primary mechanism contributing to the observed action of p32 is the ability of p32 to interact with the p53 tetramerization domain and to block p53 tetramerization, which in turn enhances nuclear export and degradation of p53, leading to defective p53 transactivation. Collectively, these data establish p32 as a negative regulator of p53 function and suggest the therapeutic potential of targeting p32 for cancer treatment.

Lithium and GADL1 regulate glycogen synthase kinase-3 activity to modulate KCTD12 expression

Potassium channel tetramerization domain containing 12 (KCTD12), the auxiliary GABAB receptor subunit, is identified as a susceptibility gene for bipolar I (BPI) disorder in the Han Chinese population. Moreover, the single-nucleotide polymorphism (SNP) rs17026688 in glutamate decarboxylase–like protein 1 (GADL1) is shown to be associated with lithium response in Han Chinese BPI patients. In this study, we demonstrated for the first time the relationship among lithium, GADL1, and KCTD12. In circulating CD11b+ macrophage cells, BPI patients showed a significantly higher percentage of KCTD12 expression than healthy controls. Among BPI patients, carriers of the ‘T’ allele (i.e., CT or TT) at site rs17026688 were found to secrete lower amounts of GADL1 but higher amounts of GABA b receptor 2 (GABBR2) in the plasma. In human SH-SY5Y neuroblastoma cells, lithium treatment increased the percentage of KCTD12 expression. Through inhibition of glycogen synthase kinase-3 (GSK-3), lithium induced cyclic AMP-response element binding protein (CREB)–mediated KCTD12 promoter activation. On the other hand, GADL1 overexpression enhanced GSK-3 activation and inhibited KCTD12 expression. We found that lithium induced, whereas GADL1 inhibited, KCTD12 expression. These findings suggested that KCTD12 may be an important gene with respect to neuron excitability and lithium response in BPI patients. Therefore, targeting GSK-3 activity and/or KCTD12 expression may constitute a possible therapeutic strategy for treating patients with BPI disorder.

Abstract 2498: MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73

Abstract MCL1 is an anti-apoptotic member of the Bcl-2 family, which has become a popular therapeutic target in cancer research over the past decade due to its propensity to promote cancer cell survival and facilitate chemoresistance. Through a phage display screen, we identified a novel MCL1 binding motif that is the reverse of the canonical BH3-binding helix that mediates interactions amongst Bcl-2 family members. Upon a BLAST sequence analysis of native proteins, we identified a putative reverse BH3 (rBH3) motif in the tumor suppressor protein, p73. Here, we show that MCL1 and p73 bind through a direct protein-protein interaction. Using fluorescence polarization assay, we characterize the strength of this interaction and reduce binding down to the rBH3-containing alpha helix in the tetramerization domain of p73. To elucidate biological function, we employ electrophoretic mobility shift assay to show that excess MCL1 negatively impacts p73 binding to DNA. Finally, using two p53-/- cancer cell lines, we demonstrate that MCL1 inhibits the transcriptional function of p73 through analysis of p73 target gene expression. In summary, we characterize a novel protein-protein interaction between MCL1 and p73 and show that MCL1 negatively impacts p73 target gene activation. This work establishes a novel function of MCL1 outside of apoptotic regulation at the mitochondria and provides new evidence of cross talk between the Bcl-2 family and the DNA damage response pathway. Citation Format: Hayley Widden, Aneta Kaczmarczyk, Robert H. Whitaker, William J. Placzek. MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2498.