Short ORFs (sORFs), that is, occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding ORFs by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural, and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/febs.15769

Revisiting sORFs: overcoming challenges to identify and characterize functional microproteins.

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

https://www.pnas.org/content/pnas/118/40/e2018899118.full.pdf

Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs

Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC.

Activation of the renin-angiotensin system, specifically in the subfornical organ is sufficient to induce fluid intake

AUG sequences and short open reading frames are commonly present in the 5′-leader sequence of G protein-coupled receptor mRNAs. The presence of these upstream AUG sequences has been demonstrated to inhibit downstream receptor translation efficiency and, most recently, receptor signal transduction. A seven amino acid peptide encoded by a short open reading frame in exon 2 of the angiotensin type 1a receptor has been shown to inhibit non-G protein-coupled signalling of angiotensin II, without altering the classical G protein-coupled pathway activated by the ligand. This finding may lead to the development of a new class of angiotensin receptor antagonists with activities biased for one, but not all, of the signalling cascades activated by angiotensin II, which could have therapeutic implications for the myriad hormones and neurotransmitters that signal through G protein-coupled receptors.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/JP270567

A 5′‐upstream short open reading frame encoded peptide regulates angiotensin type 1a receptor production and signalling via the β‐arrestin pathway

There are examples of physiological conditions under which thirst is inappropriately exaggerated, and the mechanisms for these paradoxical ingestive behaviors remain unknown. We are interested in thirst mechanisms across the female life cycle and have identified a novel mechanism through which ingestive behavior may be activated. We discovered a previously unrecognized endogenous hypothalamic peptide, phoenixin (PNX), identified physiologically relevant actions of the peptide in brain and pituitary gland to control reproductive hormone secretion in female rodents, and in the process identified the previously orphaned G protein-coupled receptor Gpr173 to be a potential receptor for the peptide. Labeled PNX binding distribution in brain parallels areas known to be important in ingestive behaviors as well in areas where gonadal steroids feedback to control estrous cyclicity (Stein LM, Tullock CW, Mathews SK, Garcia-Galiano D, Elias CF, Samson WK, Yosten GLC, Am J Physiol Regul Integr Comp Physiol 311: R489-R496, 2016). We have demonstrated upregulation of Gpr173 during puberty, fluctuations across the estrous cycle, and, importantly, upregulation during the last third of gestation. It is during this hypervolemic, hyponatremic state that both vasopressin secretion and thirst are inappropriately elevated in humans. Here, we show that central administration of PNX stimulated water drinking in both males and females under ad libitum conditions, increased water drinking after overnight fluid deprivation, and increased both water and 1.5% NaCl ingestion under fed and hydrated conditions. Importantly, losartan pretreatment blocked the effect of PNX on water drinking, and knockdown of Gpr173 by use of short interfering RNA constructs significantly attenuated water drinking in response to overnight fluid deprivation. These actions, together with the stimulatory action of PNX on vasopressin secretion, suggest that this recently discovered neuropeptide may impact the recruitment of critically important neural circuits through which ingestive behaviors and endocrine mechanisms that maintain fluid and electrolyte homeostasis are regulated.

https://journals.physiology.org/doi/pdf/10.1152/ajpregu.00023.2020

A novel regulator of thirst behavior: phoenixin.

A seven-amino acid peptide (PEP7) is encoded within a short open reading frame within exon 2 (E2) in the 5′-leader sequence (5′LS) upstream of the rat ANG 1a-receptor (rAT1aR) mRNA. A chemically synthesized PEP7 markedly inhibited ANG II-induced Erk1/2 activation in cell culture by 62% compared with a scrambled PEP7 (sPEP7) [pErk1/2/Erk1/2 (AU): ANG II, 1.000 ± 0.0, ANG II+PEP7, 0.3812 ± 0.086, ANG II+sPEP7, 1.069 ± 0.18; n = 3]. Under these same conditions, PEP7 had no effect on ANG II-stimulated inositol-trisphosphate production. PEP7 also had no effect on epidermal growth factor- and phorbol methyl ester-induced Erk1/2 activation, suggesting PEP7 selectively inhibits AT1aR-mediated Erk1/2 signaling. PEP7 intracerebroventricularly inhibited ANG II-induced saline intake but had no effect on water intake in male and female rats, indicating PEP7 also selectively inhibits the ANG II-Erk1/2 pathway in vivo since saline drinking is Erk1/2-mediated, while water drinking is not. PEP7 inhibition of ANG II-induced saline ingestion was rapidly reversed by a subsequent intracerebroventricular injection of an oxytocin antagonist, suggesting when PEP7 blocks ANG II-stimulated Erk1/2 activation, animals no longer ingest saline to balance the continued water intake, due to the release of oxytocin and its subsequent inhibitory effects on saline drinking. PEP7 also attenuated ANG II-induced increases in arterial pressure by 35% compared with sPEP7 at the same dose. Thus, we have identified a novel peptide encoded within the rAT1aR E2 that selectively inhibits Erk1/2 activation, resulting in physiological consequences for sodium ingestion and arterial pressure that may have implications for treating sodium-sensitive diseases like hypertension and chronic kidney disease.

Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide

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Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide