AP-64, Encoded by C5orf46, Exhibits Antimicrobial Activity against Gram-Negative Bacteria.
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The gene product is identified as an anionic, cysteine-free antimicrobial peptide of 64 residues - which is exactly UniProt's annotated mature chain 24..87 of Q6UWT4.
"AP-64 is an anionic amphiphilic peptide lacking cysteines (MW = 7.2, PI = 4.54)"
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Purified peptide kills four Gram-negative species. This is the primary activity measurement.
"AP-64 exhibited significant antibacterial activity against Gram-negative bacteria, including Escherichia coli DH5α, Escherichia coli O157:H7, Vibrio cholerae, and Pseudomonas aeruginosa."
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The Gram-negative specificity is measured in both arms: the two Gram-positive pathogens tested are unaffected. This internal negative control is what licenses the specific GO term rather than its parent.
"AP-64 and Gm94 lacked appreciable bactericidal activity against the pathogenic Gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes"
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Yeast is also unaffected, so the activity is not general to microbes.
"we also observed that the yeast cells were unsusceptible to the peptide treatment"
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Killing is concentration-dependent over two orders of magnitude, not an all-or-nothing observation at one dose.
"AP-64 and Gm94 effectively inhibited the growth of bacteria in the concentration range of 0.1–10 μM"
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Electron microscopy shows the bacterial cells losing shape and then disintegrating, which is the observation behind the cell-envelope reading.
"SEM examination revealed that a 2-h treatment with the proteins at the concentration of 10 μM caused an apparent irregularity and collapse in cell shape."
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The mechanism is explicitly left open between envelope damage and an intracellular target, which is why no molecular function term is proposed anywhere in this review.
"This growth curve implied that AP-64 possessed a similar antibacterial pattern to that of cell-envelope-interfering agents and that its action might be related to cell envelope damage [18]. The multiplication of growth might also hint at an intracellular target of the peptide."
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The authors state the mechanism as an open question in their own limitations, independently of the sentence above.
"Second, the molecular mechanisms underlying the bacterial inhibition should be investigated in future studies."
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Administered peptide protects mice against a lethal bacterial challenge. This is exogenous administration, so it demonstrates sufficiency and not that the endogenous gene is required.
"The survival rate of mice treated with PBS was 20% at 24 h after bacterial inoculation, whereas the survival rates of mice injected with AP-64 and Gm94 (500 μg/kg of body weight) were 90% and 80%, respectively"
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The activity is a property of the free peptide: an N-terminal SUMO fusion abolishes it and removing the tag restores it, which is a useful internal control against a preparation artefact.
"SUMO-AP-64 was expressed in a soluble form but failed to inhibit the growth of DH5α cells. After removal of the SUMO tag, AP-64 exhibited strong antibacterial effects."
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The cytotoxicity is NOT tumour-selective: normal T cells, keratinocytes and mouse embryonic fibroblasts are killed too. Any account of the Jurkat and Raji result that omits this over-states selectivity.
"Subsequently, we tested the toxicity of the peptides to T cells, Hacat, and MEF cells. Our data showed that these cells were susceptible to the peptide treatment"
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The mouse orthologue Gm94 has the same activity, so a cross-species ISS/ISO route exists in principle - but MGI currently records Gm94 as having no biological data available.
"we demonstrated that AP-64 (human C5orf46) is a type of AMP that exerts a direct antibacterial effect on Gram-negative bacteria, and our data shows that Gm94 (mouse C5orf46) also exhibits the similar antibacterial ability"
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The tissue distribution in this paper is taken from a database, not measured, and the authors say so. It is used in this review only as agreement with HPA and the HGNC alias name, never as primary evidence.
"First, the mRNA expression of AP-64 in the skin and salivary gland was discovered using the TCGA database"