CysPresso: a classification model utilizing deep learning protein representations to predict recombinant expression of cysteine-dense peptides.

BACKGROUND: Cysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence. RESULTS: We tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning. CONCLUSION: Our novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

Isoform Specificity of PKMs during Long-Term Facilitation in Aplysia Is Mediated through Stabilization by KIBRA.

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk, Aplysia californica Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix "handle" in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in AplysiaSIGNIFICANCE STATEMENT Long-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a "handle" helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms.

A role for Numb in Protein kinase M (PKM)-mediated increase in surface AMPA receptors during facilitation in Aplysia.

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over-expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin-tagged Aplysia glutamate receptor measured using live imaging after intermediate- or long-term facilitation. Over-expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate-term facilitation, but did block increases in synaptic strength seen during long-term facilitation. There was no effect of over-expression of this form of ApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long-term maintenance of synaptic strength. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

The zinc fingers of the small optic lobes calpain bind polyubiquitin.

The small optic lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al. 2017a). SOL calpains have a conserved set of 5-6 N-terminal zinc fingers. Bioinformatic analysis suggests that these zinc fingers could bind to ubiquitin. In this study, we show that both the Aplysia and mouse SOL calpain (also known as Calpain 15) zinc fingers bind ubiquitinated proteins, and we confirm that Aplysia SOL binds poly- but not mono- or diubiquitin. No specific zinc finger is required for polyubiquitin binding. Neither polyubiquitin nor calcium was sufficient to induce purified Aplysia SOL calpain to autolyse or to cleave the atypical PKC to PKM in vitro. In Aplysia, over-expression of the atypical PKC in sensory neurons leads to an activity-dependent cleavage event and an increase in nuclear ubiquitin staining. Activity-dependent cleavage is partially blocked by a dominant negative SOL calpain, but not by a dominant negative classical calpain. The cleaved PKM was stabilized by the dominant negative classical calpain and destabilized by a dominant negative form of the PKM stabilizing protein KIdney/BRAin protein. These studies provide new insight into SOL calpain's function and regulation. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Selective Erasure of Distinct Forms of Long-Term Synaptic Plasticity Underlying Different Forms of Memory in the Same Postsynaptic Neuron.

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories.

A decrease in eukaryotic elongation factor 2 phosphorylation is required for local translation of sensorin and long-term facilitation in Aplysia.

Mechanistic target of rapamycin complex 1 (mTORC1)-dependent protein synthesis is required for many forms of synaptic plasticity and memory, but the downstream pathways important for synaptic plasticity are poorly understood. Long-term facilitation (LTF) in Aplysia is a form of synaptic plasticity that is closely linked to behavioral memory and an attractive model system for examining the important downstream targets for mTORC1 in regulating synaptic plasticity. Although mTORC1-regulated protein synthesis has been strongly linked to translation initiation, translation elongation is also regulated by mTORC1 and LTF leads to an mTORC1-dependent decrease in eukaryotic elongation factor 2 (eEF2) phosphorylation. The purpose of this study is to test the hypothesis that the decrease in eEF2 phosphorylation is required for mTORC1-dependent translation and plasticity. We show that the LTF-induced decrease in eEF2 phosphorylation is blocked by expression of an eEF2 kinase (eEF2K) modified to be resistant to mTORC1 regulation. We found that expression of this modified kinase blocked LTF. LTF requires local protein synthesis of the neuropeptide sensorin and importantly, local sensorin synthesis can be measured using a dendra fluorescent protein containing the 5' and 3' untranslated regions (UTRs) of sensorin. Using this construct, we show that blocking eEF2 dephosphorylation also blocks the increase in local sensorin synthesis. These results identify decreases in eEF2 phosphorylation as a critical downstream effector of mTOR required for long-term plasticity and identify an important translational target regulated by decreases in eEF2 phosphorylation.

Investigating the Potential Signaling Pathways That Regulate Activation of the Novel PKC Downstream of Serotonin in Aplysia.

Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT) underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF) receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF)-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs) or other molecules must also be involved in translocation of PKC Apl II.

Clozapine and therapeutic drug monitoring: is there sufficient evidence for an upper threshold?

RATIONALE: Clozapine levels are advocated in the monitoring of patients on this drug and have now been used for a number of years. A safety-related threshold has also been proposed, as well as therapeutic lower and upper thresholds. While there has been reasonable consensus regarding a lower therapeutic threshold, this is not the case for the upper thresholds. OBJECTIVES: Our aim was to review available evidence related to upper thresholds. METHODS: We carried out an electronic search of different databases and a manual search of articles between 1960 and 2011, cross-referencing the following terms with clozapine-interactions, monitoring, pharmacokinetics, plasma levels, serum levels, and toxicity. RESULTS: Sixty-nine articles met our search criteria and these could be divided into reviews (11), studies (24), and case reports (35). Study quality was evaluated, and none met criteria for a prospective, randomized controlled trial specifically addressing higher plasma levels, e.g., >500 ng/ml. Case reports emphasize in particular the impact of interactions, e.g., antidepressants and smoking. There is clear evidence indicating a dose-related increased risk of seizures, at least to 500-600 mg/day, but a lack of data to suggest such a relationship between plasma levels, dose, and side effects linked to safety, e.g., seizures, myocarditis, and agranulocytosis. The very limited evidence addressing an upper threshold related to clinical response suggests a "ceiling effect" in the range of 600-838 ng/ml. CONCLUSIONS: It appears that the current safety-related threshold is not supported by evidence. There may be an upper threshold for clinical response, beyond which chance of response falls off, although further studies are warranted.