Linkage between Psychological Factors and Response to Immune Checkpoint Inhibitor Therapy: A Preliminary Study.

Substantial evidence has accumulated showing that psychological distress affects immune regulation, the response to cancer treatment, and survival. The effect of psychological parameters on the effectiveness of immune checkpoint inhibitor (ICI) treatment has not yet been studied. This preliminary study aimed to (a) examine the associations between psychological factors and responses to ICI treatment and (b) assess the associations between psychological factors and blood measures of sPD-1, sCTLA-4, and cytokines that may alter the effect of ICI treatment. The participants were 62 individuals with advanced cancer, aged 18 years or older, who were candidates for ICI treatment as a new line of treatment. The participants answered questionnaires and provided blood samples and medical data prior to the start of ICI treatment and 3 months after. Perceived health status was positively associated with better responses to ICI treatment. In the subsample of participants with biomarkers, worse health-related quality of life was associated with higher IL-6 and sCTLA-4; emotional distress and sleep difficulties were associated with higher sCTLA-4; and better perceived health was associated with lower IL-6 and TNFα. sPD-1 was not associated with psychological measures. This preliminary study found for the first time that some psychological measures could be linked to responses to cancer treatment, possibly via pro-inflammatory cytokines and sCTLA-4.

Fear Conditioning Leads to Enduring Alterations in RNA Transcripts in Hippocampal Neuropil that are Dependent on EphB2 Forward Signaling.

Alterations in mRNA transcription have been associated with changes in brain functions. We wanted to examine if fear conditioning causes long-term changes in transcriptome profiles in the basolateral amygdala (BLA) and hippocampus using RNA-Seq and laser microdissection microscopy. We further aimed to uncover whether these changes are involved in memory formation by monitoring their levels in EphB2lacZ/lacZ mice, which lack EphB2 forward signaling and can form short-term fear conditioning memory but not long-term fear conditioning memory. We found transcriptome signatures unique to each brain region that are comprise of specific cellular pathways. We also revealed that fear conditioning leads to alterations in mRNAs levels 24 h after training in hippocampal neuropil, but not in hippocampal cell layers or BLA. The two main groups of altered mRNAs encode proteins involved in neuronal transmission, neuronal morphogenesis and neuronal development and the vast majority are known to be enriched in neurons. None of these mRNAs levels were altered by fear conditioning in EphB2lacZ/lacZ mice, which were also impaired in long-term fear memory. We show here that fear conditioning leads to an enduring alteration in mRNAs levels in hippocampal neuropil that is dependent on processes mediated by EphB2 that are needed for long-term memory formation.

Rac1 GTPase activation impairs fear conditioning-induced structural changes in basolateral amygdala neurons and long-term fear memory formation.

Long-term memory formation leads to enduring alterations in synaptic efficacy and neuronal responses that may be created by changes in neuronal morphology. We show that fear conditioning leads to a long-lasting increase in the volume of the primary and secondary dendritic branches, but not of distal branches, of neurons located at the basolateral amygdala (BLA). The length of the dendritic branches is not affected by fear conditioning. Fear conditioning leads to an enduring increase in the length and volume of dendritic spines, especially in the length of the spine neck and the volume of the spine head. Fear conditioning does not affect dendritic spine density. We further reveal that activation of Rac1 in BLA during fear conditioning impairs long-term auditory, but not contextual, fear conditioning memory. Activation of Rac1 during fear conditioning prevents the enduring increase in the dendritic primary branch volume and dendritic spines length and volume. Rac1 activation per se has no effect on neuronal morphology. These results show that fear conditioning induces changes known to reduce the inhibition of signal propagation along the dendrite and the increase in synaptic efficacy whereas preventing these changes, by Rac1 activation, impairs fear memory formation.

Nck1 activity in lateral amygdala regulates long-term fear memory formation.

Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathological conditions such as phobias and post-traumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). Nck1 is a key neuronal adaptor protein involved in the regulation of the actin cytoskeleton and the neuronal processes believed to be involved in memory formation. However, the role of Nck1 in memory formation is not known. Here we explored the role of Nck1 in fear memory formation in lateral amygdala (LA). Reduction of Nck1 in excitatory neurons in LA enhanced long-term, but not short-term, auditory fear conditioning memory. Activation of Nck1, by using a photoactivatable Nck1 (PA-Nck1), during auditory fear conditioning in excitatory neurons in LA impaired long-term, but not short-term, fear memory. Activation of Nck1 immediately or a day after fear conditioning did not affect fear memory. The hippocampal-mediated contextual fear memory was not affected by the reduction or activation of Nck1 in LA. We show that Nck1 is localized to the presynapses in LA. Nck1 activation in LA excitatory neurons decreased the frequency of AMPA receptors-mediated miniature excitatory synaptic currents (mEPSCs). Nck1 activation did not affect GABA receptor-mediated inhibitory synaptic currents (mIPSCs). These results show that Nck1 activity in excitatory neurons in LA regulates glutamate release and sets the threshold for fear memory formation. Moreover, our research shows that Nck1 may serve as a target for pharmacological treatment of fear and anxiety disorders.

EphrinA4 mimetic peptide impairs fear conditioning memory reconsolidation in lateral amygdala.

Fear memory may undergo a process after memory reactivation called reconsolidation. To examine the roles of ephrinA4 in fear memory reconsolidation an inhibitory ephrinA4 mimetic peptide (pep-ephrinA4), that targets the EphA binding site and inhibits EphA activation, was used. Pep-ephrinA4 was microinjected into the lateral amygdala (LA) of fear-conditioned rats 24 h after training and 30 min before tone CS memory retrieval. Memory retrieval was unaffected by pep-ephrinA4. However, the animals were impaired in fear memory tested 1 h or 24 h afterward when compared to controls. Fear-conditioned animals injected with pep-ephrinA4 into LA immediately after long-term memory retrieval were unaffected when tested 24 h afterward. Microinjection into LA of a peptide originated from an ephrinA4 site that does not interact with EphA did not affect fear memory reconsolidation. Rats that were administrated with pep-ephrinA4 systemically 24 h after fear conditioning and 30 min before CS memory retrieval were impaired in long-term fear conditioning memory tested 24 h afterward when compared to the control peptide. These results show that ephrinA4 binding sites are needed for long-term fear memory reconsolidation in LA and may serve as a target for the treatment of fear-related disorders by blocking reconsolidation.

In Silico Structural and Biochemical Functional Analysis of a Novel CYP21A2 Pathogenic Variant.

Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694-696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1-4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects.

The Role of Rac GTPase in Dendritic Spine Morphogenesis and Memory.

The ability to form memories in the brain is needed for daily functions, and its impairment is associated with human mental disorders. Evidence indicates that long-term memory (LTM)-related processes such as its consolidation, extinction and forgetting involve changes of synaptic efficacy produced by alterations in neural transmission and morphology. Modulation of the morphology and number of dendritic spines has been proposed to contribute to changes in neuronal transmission mediating such LTM-related processes. Rac GTPase activity is regulated by synaptic activation and it can affect spine morphology by controlling actin-regulatory proteins. Recent evidence shows that changes in Rac GTPase activity affect memory consolidation, extinction, erasure and forgetting and can affect spine morphology in brain areas that mediate these behaviors. Altered Rac GTPase activity is associated with abnormal spine morphology and brain disorders. By affecting Rac GTPase activity we can further understand the roles of spine morphogenesis in memory. Moreover, manipulation of Rac GTPase activity may serve as a therapeutic tool for the treatment of memory-related brain diseases.

Long-term memory is maintained by continuous activity of Arp2/3 in lateral amygdala.

Evidence indicates that long-term memory formation involves alterations in synaptic efficacy produced by modifications in neural transmission and morphology. However, it is not clear how such changes induced by learning, that encode memory, are maintained over long period of time to preserve long-term memory. It has been shown that the actin nucleating protein Arp2/3 is essential for supporting neuronal morphology and synaptic transmission. We therefore hypothesized that continuous Arp2/3 activity is needed to maintain long-term memory over time. To test this hypothesis we microinjected into lateral amygdala (LA) of rats CK-666, a specific inhibitor of Arp2/3, two days after fear conditioning and tested the effect on long-term fear memory maintenance a day afterward. We found that injection of CK-666 two days after training abolished fear conditioning memory. Fear conditioning could be formed when a control compound CK-689 was applied two days after training. Microinjection of CK-666 a day before fear conditioning training had no effect on fear conditioning learning and long-term memory formation. We revealed that Arp2/3 is also needed to maintain long-term conditioned taste aversion (CTA) memory in LA. Microinjection of CK-666 two days after CTA training impaired long-term memory tested a day afterwards. We conclude that continuous activity of Arp2/3 in LA is essential for the maintenance of long-term memory.

EphB2 receptor forward signaling is needed for normal long-term memory formation in aged mice.

The molecular mechanisms underpinning age-related changes in the ability to form long-term memory need to be clarified. EphB2 receptors and their ephrin ligands are involved in key cellular functions such as neuronal morphogenesis and synaptic transmission believed to be involved in long-term memory formation. We were therefore interested to explore whether EphB2 is involved in the alterations in memory formation abilities observed in old age. Toward that end, we examined the ability to form long-term memory in mice that lack EphB2 (EphB2-/-). A previous study has shown that the ability to form long-term conditioned taste aversion (CTA) memory in young EphB2-/- mice remains intact. In the present study, we report that long-term CTA memory formation is improved in old wild-type mice but not in age-matched old EphB2-/- mice. To further explore EphB2 mechanisms responsible for this difference in memory formation ability, we examined CTA memory in EphB2lacZ/lacZ mice devoid of EphB2 forward signaling. We found that the ability to create CTA long-term memory is unaffected in young EphB2lacZ/lacZ mice. However, the ability to form an increased long-term CTA memory shown in old wild-type mice is impaired in old EphB2lacZ/lacZ mice. The inability to form enhanced CTA long-term memory in EphB2-/- and EphB2lacZ/lacZ old mice was not caused by differences in taste perception or ability to consume fluids. Thus, our observations show that the absence of EphB2 forward signaling in old mice impairs the ability to form enhanced long-term CTA memory and indicate that EphB2 forward signaling is needed for normal memory formation in aged mice.

Possible cooption of a VEGF-driven tubulogenesis program for biomineralization in echinoderms.

Biomineralization is the process by which living organisms use minerals to form hard structures that protect and support them. Biomineralization is believed to have evolved rapidly and independently in different phyla utilizing preexisting components. The mechanistic understanding of the regulatory networks that drive biomineralization and their evolution is far from clear. Sea urchin skeletogenesis is an excellent model system for studying both gene regulation and mineral uptake and deposition. The sea urchin calcite spicules are formed within a tubular cavity generated by the skeletogenic cells controlled by vascular endothelial growth factor (VEGF) signaling. The VEGF pathway is essential for biomineralization in echinoderms, while in many other phyla, across metazoans, it controls tubulogenesis and vascularization. Despite the critical role of VEGF signaling in sea urchin spiculogenesis, the downstream program it activates was largely unknown. Here we study the cellular and molecular machinery activated by the VEGF pathway during sea urchin spiculogenesis and reveal multiple parallels to the regulation of vertebrate vascularization. Human VEGF rescues sea urchin VEGF knockdown, vesicle deposition into an internal cavity plays a significant role in both systems, and sea urchin VEGF signaling activates hundreds of genes, including biomineralization and interestingly, vascularization genes. Moreover, five upstream transcription factors and three signaling genes that drive spiculogenesis are homologous to vertebrate factors that control vascularization. Overall, our findings suggest that sea urchin spiculogenesis and vertebrate vascularization diverged from a common ancestral tubulogenesis program, broadly adapted for vascularization and specifically coopted for biomineralization in the echinoderm phylum.

Biophysical and structural characterization of the small heat shock protein HspA from Thermosynechococcus vulcanus in 2 M urea.

Small heat shock proteins (sHSPs) belong to the superfamily of molecular chaperones. They prevent aggregation of partially unfolded or misfolded client proteins, providing protection to organisms under stress conditions. Here, we report the biophysical and structural characterization of a small heat shock protein (HspA) from a thermophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 M urea. HspA has been shown to be important for the protection of Photosystem II and the Phycobilisome antenna complex at elevated temperatures. Heterologously expressed HspA requires the presence of 1-2 M urea to maintain its solubility at concentrations required for most characterization methods. Spectroscopic studies reveal the presence of the ß-sheet structure and intactness of the tertiary fold in HspA. In vitro assays show that the HspA maintains chaperone-like activity in protecting soluble proteins from thermal aggregation. Chromatography and electron microscopy show that the HspA exists as a mixture of oligomeric forms in the presence of 2 M urea. HspA was successfully crystallized only in the presence of 2 M urea. The crystal structure of HspA shows urea-induced loss of about 30% of the secondary structure without major alteration in the tertiary structure of the protein. The electron density maps reveal changes in the hydrogen bonding network which we attribute to the presence of urea. The crystal structure of HspA demonstrates a mixture of both direct interactions between urea and protein functionalities and interactions between urea and the surrounding solvent that indirectly affect the protein, which are in accordance with previously published studies.

Activation of EphB2 Forward Signaling Enhances Memory Consolidation.

EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca2+ responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2lacZ/lacZ). Activation of optoEphB2 in LA of EphB2lacZ/lacZ mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.

Affecting long-term fear memory formation through optical control of Rac1 GTPase and PAK activity in lateral amygdala.

Fear conditioning, a behavioral model for studying fear-related disorders, is believed to be formed by alterations of synaptic efficacy mediated by changes in synaptic transmission and neuronal morphology in lateral amygdala (LA). Rac GTPase and its downstream effector p21-activated kinase (PAK) are involved in such key neuronal functions. Here we show that optical activation of Rac1 GTPase using photoactivatable form of Rac1 (PA-Rac1) in amygdala led to phosphorylation of PAK and inhibition of long-term but not short-term auditory fear conditioning memory formation. Activation of PA-Rac1 in LA one day after fear conditioning had no effect on long-term fear memory tested 24 hrs after PA-Rac1 activation. Inhibition of PAK in LA by microinjection of the PAK inhibitor IPA-3 30 minutes before fear conditioning enhanced long-term but not short-term fear memory formation. Our results demonstrate that photoactivation of Rac1 GTPase in lateral amygdala impairs fear memory formation. Moreover, Rac1 effector PAK activity during fear conditioning constrains the formation of fear memory in LA. Thus, Rac GTPase and PAK proteins may serve as targets for treatment of fear and anxiety disorders.

The Role of Ephs and Ephrins in Memory Formation.

The ability to efficiently store memories in the brain is a fundamental process and its impairment is associated with multiple human mental disorders. Evidence indicates that long-term memory formation involves alterations of synaptic efficacy produced by modifications in neural transmission and morphology. The Eph receptors and their cognate ephrin ligands have been shown to be involved in these key neuronal processes by regulating events such as presynaptic transmitter release, postsynaptic glutamate receptor conductance and trafficking, synaptic glutamate reuptake, and dendritic spine morphogenesis. Recent findings show that Ephs and ephrins are needed for memory formation in different organisms. These proteins participate in the formation of various types of memories that are subserved by different neurons and brain regions. Ephs and ephrins are involved in brain disorders and diseases with memory impairment symptoms, including Alzheimer's disease and anxiety. Drugs that agonize or antagonize Ephs/ephrins signaling have been developed and could serve as therapeutic agents to treat such diseases. Ephs and ephrins may therefore induce cellular alterations mandatory for memory formation and serve as a target for pharmacological intervention for treatment of memory-related brain diseases.

The roles of Eph receptors in contextual fear conditioning memory formation.

Eph receptors regulate glutamate receptors functions, neuronal morphology and synaptic plasticity, cellular events believed to be involved in memory formation. In this study we aim to explore the roles of Eph receptors in learning and memory. Toward that end, we examined the roles of EphB2 and EphA4 receptors, key regulators of synaptic functions, in fear conditioning memory formation. We show that mice lacking EphB2 (EphB2(-/-)) are impaired in short- and long-term contextual fear conditioning memory. Mice that express a carboxy-terminally truncated form of EphB2 that lacks forward signaling, instead of the full EphB2, are impaired in long-term, but not short-term, contextual fear conditioning memory. Long-term contextual fear conditioning memory is attenuated in CaMKII-cre;EphA4(lx/-) mice where EphA4 is removed from all pyramidal neurons of the forebrain. Mutant mice with targeted kinase-dead EphA4 (EphA4(KD)) exhibit intact long-term contextual fear conditioning memory showing that EphA4 kinase-mediated forward signaling is not needed for contextual fear memory formation. The ability to form long-term conditioned taste aversion (CTA) memory is not impaired in the EphB2(-/-) and CaMKII-cre;EphA4(lx/-) mice. We conclude that EphB2 forward signaling is required for long-term contextual fear conditioning memory formation. In contrast, EphB2 mediates short-term contextual fear conditioning memory formation in a forward signaling-independent manner. EphA4 mediates long-term contextual fear conditioning memory formation in a kinase-independent manner.

The Membrane Proximal Region of AMPA Receptors in Lateral Amygdala is Essential for Fear Memory Formation.

The membrane proximal region (MPR) of AMPA receptor (AMPAR) is needed for receptor trafficking and synaptic plasticity. However, its roles in long-term memory formation are not known. To assess the possible roles of AMPAR-MPR in rat lateral amygdala (LA) in short- and long-term fear memory formation, we used glutamate receptors (GluAs)-MPR competitive peptides MPR(DD) and MPR(AA). The MPR(DD) peptide is derived from GluA1 MPR and was previously shown to impair synaptic plasticity and to inhibit GluA1 containing AMPAR insertion into the synapse in an activity-dependent manner. The MPR(AA) peptide is derived from GluA2/4 MPR, and this receptor fragment was shown to be essential for GluA4 protein interaction needed for its insertion into the neuronal membrane and synapse. The peptides were linked to a TAT peptide (TAT-MPR(DD) and TAT-MPR(AA)) to facilitate internalization into LA cells. Infusion of the TAT-MPR(DD) peptide into LA 30 min before fear conditioning led to a significant impairment of long-term fear memory formation. Injection of TAT-MPR(DD) peptide into LA 30 min before fear conditioning impaired short-term fear memory formation. The TAT-MPR(DD) peptide had no effect on memory retrieval when injected into LA 30 min before fear memory test. Infusion of the TAT-MPR(AA) peptide into LA 30 min before fear conditioning led to a significant impairment of long-term fear memory formation. In contrast, the TAT-MPR(AA) had no effect on short-term fear memory formation. A TAT-control peptide had no effect on short- or long-term fear memory. These results show that the AMPAR-MPR in LA is needed for fear memory formation and that the MPR region of GluA1 is essential for acquisition of memory, whereas the MPR region of GluA4 is essential for long-term fear memory consolidation.

Acetylation represses the binding of CheY to its target proteins.

The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications - phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non-enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM - its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheY's ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.

A two-state electronic antigen and an antibody selected to discriminate between these states.

Inspired by biology where pathways are triggered and suppressed by specific binding of two molecules, we realize a functional interface between electronics and biology by replacing one of the pair molecules with a two-state "electronic antigen" device comprising a hydroquinone monolayer assembled on gold, and choosing for the pair molecule an antibody that discriminates between the two electrically selected redox states of the monolayer. Application of an oxidative +0.6 V pulse to the antigen switches it to its benzoquinone state where antibodies bind the layer. A subsequent -0.6 V pulse reduces the monolayer back to the unbinding hydroquinone state, releases the specifically bound antibody molecules, and prevents further binding.

Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome.

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (approximately 6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via "structural mimicry" due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.

Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2(Ank-SH3)) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2(Ank-SH3) binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2(Ank-SH3) to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2(Ank-SH3) to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2(Ank-SH3) with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2(Ank-SH3) with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.