Nadia Milech

The Neuroprotective Efficacy of Cell-Penetrating Peptides TAT, Penetratin, Arg-9, and Pep-1 in Glutamic Acid, Kainic Acid, and In Vitro Ischemia Injury Models Using Primary Cortical Neuronal Cultures

Cell-penetrating peptides (CPPs) are small peptides (typically 5–25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen–glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9xa0μM) and kainic acid (IC50: 0.81, 2.0, 6.2xa0μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0xa0μM, TAT-D: 7.1xa0μM, penetratin/Pep-1: >10xa0μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15xa0min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to deliver neuroprotective drugs to the CNS following injury and/or potential neuroprotectants in their own right.

Novel alternative PBX3 isoforms in leukemia cells with distinct interaction specificities

PBX3 is a member of the PBX family of TALE homeobox genes. The prototypic member, PBX1, was first identified in chromosomal translocations in B‐lineage leukemia and is required for normal hematopoiesis. PBX2 and PBX3 were later identified as members of this highly conserved family by their strong homology to PBX1. While the expression pattern of PBX1 is restricted, PBX2 and PBX3 are ubiquitously expressed. Little is known about the functional role of PBX3. Our studies identified two PBX3 transcripts alternative to the canonical forms, PBX3A and PBX3B, resulting from a novel splice in PBX3. These new isoforms, named PBX3C and PBX3D, have been detected in all tissues and cell lines tested. Intriguingly, expression of PBX3D is favored in normal cells, whereas PBX3C expression is favored in leukemia cells. Functional studies showed that PBX3C and PBX3D proteins were unable to interact with the PBX‐interacting factor PREP1 and weakly interacted with MEIS proteins. We propose that PBX3C and PBX3D may affect PBX3‐mediated transcriptional regulation by acting in opposition to the known PBX proteins through alternative PBX3 complex formation. The identification and characterization of these novel PBX3 isoforms provide a foundation for a better understanding of the biological role of PBX3.

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer ® peptides

Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5 monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; Pu2009<u20090.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function.

MEIS proteins as partners of the TLX1/HOX11 oncoprotein

Aberrant expression of the TLX1/HOX11 proto-oncogene is associated with a significant subset of T-cell acute lymphoblastic leukemias (T-ALL). Yet the manner in which TLX1 contributes to oncogenesis is not fully understood. Since, typically, interactions of HOX and TALE homeodomain proteins are determinant of HOX function, and HOX/MEIS co-expression has been shown to accelerate some leukemias, we systematically examined whether TLX1 interacts with MEIS and PBX proteins. Here, we report that TLX1 and MEIS proteins both interact and are co-expressed in T-ALL, and suggest that co-operation between TLX1 and MEIS proteins may have a significant role in T-cell leukemogenesis.

CHAPTER 17. Phylomer Libraries: A Rich Source of Peptide Hits in Phenotypic and Target-Directed Screens

Phylomers are peptides derived from fragments of proteins encoded by biodiverse genomes. Phylomer libraries were devised as a strategy to enable screening of a diverse array of peptides representing sequences that form secondary and super-secondary structures within natural proteins, some of which have evolved to bind to protein interfaces. By screening biodiverse peptide libraries, we aim to overcome poor hit rates in phenotypic and target-directed screens of random peptide libraries, presumed to be due to the limited structural complexity that can be assumed by the random amino acid composition of these peptides. Consistent with this strategy, we present evidence that Phylomer libraries derived from biodiverse genomes do offer a rich source of high-quality hits in both target-directed and phenotypic screens. We present a bioinformatic analysis of Phylomer peptide libraries and empirical data from representative case studies of Phylomer library screens. Taken together, these data illustrate the utility of this alternative approach to identify peptide hits of high quantity and quality, both for therapeutic applications and as probes for target identification and validation.