Beyond keratinocyte differentiation: emerging new biology of small proline-rich proteins

Abstract

Small proline-rich proteins (SPRRPs) are traditionally known for their function in keratinocyte homeostasis. Recent evidence demonstrates their involvement in additional diverse physiological processes ranging from p53 signaling and direct prevention of DNA damage to bactericidal activities. We highlight these novel, intriguing roles of SPRRPs and discuss them in the context of relevant pathological conditions. Small proline-rich proteins (SPRRPs) are traditionally known for their function in keratinocyte homeostasis. Recent evidence demonstrates their involvement in additional diverse physiological processes ranging from p53 signaling and direct prevention of DNA damage to bactericidal activities. We highlight these novel, intriguing roles of SPRRPs and discuss them in the context of relevant pathological conditions. The multigene family of SPRRPs consists of hitherto identified 11 structurally highly homologous members: SPRR1A, SPRR1B, SPRR2A-G, SPRR3, and SPRR4. One of the most extensively studied biological functions of SPRRPs is their role in the formation of the cornified cell envelope (CE) (see Glossary) in keratinocytes. SPRRPs’ activity becomes vital during later phases of keratinocyte differentiation, when their glutamine- and lysine-rich N and C termini are cross-linked with loricrin by calcium-dependent transglutaminases 1 and 3, resulting in the formation of an insoluble, highly protective CE (Figure 1A ). SPRRPs dictate the CE’s toughness, strength, and flexibility, characteristic of stratified squamous epithelia [1.Cabral A. et al.Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function.J. Biol. Chem. 2001; 276: 19231-19237Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. The property that sets SPRRPs apart from other CE components is their highly repetitive proline-rich central domain, characterized by a variable consensus sequence between the different family members. However, recent findings suggest that SPRRPs are involved in additional pleiotropic biological functions that go beyond their established role in keratinocyte homeostasis. SPRRPs have been shown to facilitate cell migration and wound healing in keratinocytes [2.Vermeij W.P. Backendorf C. Skin cornification proteins provide global link between ROS detoxification and cell migration during wound healing.PLoS One. 2010; 5e11957Crossref Scopus (67) Google Scholar], induce epithelial–mesenchymal transition in cholangiocytes [3.Mizuguchi Y. et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.BMC Mol. Biol. 2012; 13: 20Crossref PubMed Scopus (29) Google Scholar,4.Specht S. et al.SPRR2A expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis.Clin. Exp. Metastasis. 2013; 30: 877-890Crossref PubMed Scopus (14) Google Scholar], and alter invasiveness and metastatic capabilities in cholangiocarcinoma models [4.Specht S. et al.SPRR2A expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis.Clin. Exp. Metastasis. 2013; 30: 877-890Crossref PubMed Scopus (14) Google Scholar] (Figure 1B). Intriguingly, SPRRPs play important roles in free radical quenching [2.Vermeij W.P. Backendorf C. Skin cornification proteins provide global link between ROS detoxification and cell migration during wound healing.PLoS One. 2010; 5e11957Crossref Scopus (67) Google Scholar,5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar, 6.Huynh K.M. et al.Sprr2f protects against renal injury by decreasing the level of reactive oxygen species in female mice.Am. J. Physiol. Ren. Physiol. 2020; 319: F876-F884Crossref PubMed Google Scholar, 7.Pradervand S. et al.Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.EMBO J. 2004; 23: 4517-4525Crossref PubMed Scopus (79) Google Scholar], direct prevention of chromosomal damage [5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar], and p53 signaling [3.Mizuguchi Y. et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.BMC Mol. Biol. 2012; 13: 20Crossref PubMed Scopus (29) Google Scholar,8.Burke R.M. et al.Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3436-E3445Crossref PubMed Scopus (26) Google Scholar], linking them to the pathogenesis of many diseases, including fibrosis-associated heart failure and cancer [8.Burke R.M. et al.Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3436-E3445Crossref PubMed Scopus (26) Google Scholar, 9.Xu X. et al.Serum small proline-rich protein 2A (SPRR2A) is a noninvasive biomarker in gastric cancer.Dis. Markers. 2020; 8493796Crossref Scopus (4) Google Scholar, 10.Nisa L. et al.Comprehensive genomic profiling of patient-matched head and neck cancer cells: a preclinical pipeline for metastatic and recurrent disease.Mol. Cancer Res. 2018; 16: 1912-1926Crossref PubMed Scopus (19) Google Scholar]. A testament to the family’s remarkable versatility is the recent identification of SPRR2A as a novel antimicrobial protein capable of selectively killing Gram-positive gut bacteria by disrupting their cell membranes [11.Hu Z. et al.Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.Science. 2021; 374eabe6723Crossref Scopus (24) Google Scholar]. Similarly, SPRR1A and SPRR2A are active against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, thus supporting the formation of cutaneous barrier that defends the host against systemic infection [12.Zhang C. et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.Elife. 2022; 11e76729Crossref Scopus (12) Google Scholar]. In addition to providing protection against mechanical, chemical, and biological noxae as part of the CE in squamous tissues, SPRRPs can directly quench reactive oxygen species (ROS) via oxidation of cysteine residues abundant in their primary structures (Figure 1C). The importance of cysteine in protection from oxidative stress was confirmed through chemical modifications of its residues, which resulted in an almost complete loss of SPRR1B’s ROS-quenching capacity [2.Vermeij W.P. Backendorf C. Skin cornification proteins provide global link between ROS detoxification and cell migration during wound healing.PLoS One. 2010; 5e11957Crossref Scopus (67) Google Scholar,5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar]. Cysteine oxidation triggers the formation of SPRRP multimers, whereby SPRRPs spontaneously associate into dimers, trimers, and tetramers via the formation of cysteine disulfide bonds, thus giving a rise to a large cytoplasmic structure referred to as a cellular antioxidant shield [5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar]. Furthermore, SPRRPs bind directly to DNA to prevent strand breaks upon exposure to ROS. Both quenching of ROS via cysteine oxidation and protection from DNA strand breaks via direct DNA binding are tightly regulated by the oxidation state of SPRRPs [5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar]. Although members of all four classes of SPRRPs possess the ability to bind DNA, SPRR4 required ten times lower molar quantities to achieve comparable DNA binding relative to SPRR1-3 class proteins [5.Vermeij W.P. et al.Proteomic identification of in vivo interactors reveals novel function of skin cornification proteins.J. Proteome Res. 2012; 11: 3068-3076Crossref PubMed Scopus (21) Google Scholar]. The importance of SPRRPs in defense against ROS in renal cells was demonstrated in a ureteral obstruction mouse model, where SPRR2F was among the most upregulated genes following renal injury. SPRR2F-knockout mice exhibited greater renal damage following unilateral ureteral obstruction compared to wild-type animals. This was validated by a significant decrease in cell viability upon ROS induction in renal epithelial cells derived from SPRR2F-knockout animals, which could be rescued by glutathione supplementation [6.Huynh K.M. et al.Sprr2f protects against renal injury by decreasing the level of reactive oxygen species in female mice.Am. J. Physiol. Ren. Physiol. 2020; 319: F876-F884Crossref PubMed Google Scholar]. Similar protective effects were observed in cardiac cells. In vitro models of ischemic stress featured less structural damage and markedly reduced rates of apoptosis in rat cardiomyocytes overexpressing SPRR1A. A significantly higher tolerance to ischemic stress and absence of markers related to global cardiac injury were confirmed in vivo in SPRR1A-overexpressing murine cardiomyocytes. In these models, SPRR1A accumulated along myofibrils, where it engaged in ROS quenching to protect cardiomyocytes from ROS-mediated damage [7.Pradervand S. et al.Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.EMBO J. 2004; 23: 4517-4525Crossref PubMed Scopus (79) Google Scholar]. The observation that some processes stimulated by SPRR2A overexpression – such as increased cell viability upon ROS exposure, and acquisition of mesenchymal properties – may be impeded by p53-induced cell senescence or death under harmful conditions led to the hypothesis that SPRR2A may need to suppress p53 activity to exert these effects. Indeed, it has since been shown [3.Mizuguchi Y. et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.BMC Mol. Biol. 2012; 13: 20Crossref PubMed Scopus (29) Google Scholar] that SPRR2A affects p53 stability through its deacetylation. This is accomplished via two distinct pathways: first, SPRR2A interferes with p300–p53 binding and diminishes p300-mediated transfer of acetyl groups onto p53 by complexing with the CH3 domain of p300 through its xPxxP motifs, and second, SPRR2A increases expression of histone deacetylase 1 (HDAC1), resulting in direct HDAC1-mediated removal of acetyl groups from p53. Additionally, by competing with p53 for binding to p300, HDAC1 can interfere with p300-mediated p53 acetylation. K382 acetylation of p53 stabilizes the protein and leads to its accumulation, allowing p53 to exert its transcriptional activity on many cellular promoters and leading, for example, to enhanced p21 expression [3.Mizuguchi Y. et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.BMC Mol. Biol. 2012; 13: 20Crossref PubMed Scopus (29) Google Scholar]. p21 promoter activity is significantly reduced in SPRR2A-overexpressing cholangiocarcinoma cells, the decrease in p53 acetylation being related to a reduction in expression of its downstream effectors [3.Mizuguchi Y. et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.BMC Mol. Biol. 2012; 13: 20Crossref PubMed Scopus (29) Google Scholar]. Interestingly, a separate mechanism that links one of the SPRRPs to p53 stability has been described recently [8.Burke R.M. et al.Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3436-E3445Crossref PubMed Scopus (26) Google Scholar] (Figure 1D). In a mouse model of congestive heart failure (CHF), SPRR2B expression activated fibroblasts and increased their proliferation, specifically upon exposure to transforming growth factor β and oxidative stress, two of the known stimuli of cardiac pathological remodeling. The underlying process proved to be SPRR2B binding to ubiquitin-specific protease 7 (USP7), a ubiquitin ligase, which interacts with and deubiquitinates mouse double minute 2 homolog (MDM2). Formation of the SPRR2B–USP7 complex leads to increased removal of ubiquitin residues from MDM2, which allows MDM2 interaction with p53 and consequently promotes its ubiquitination and degradation. In CHF, these events attenuate apoptosis and increase the expression of several cyclins, such as CDK1, CCA2K or CCNB1, thus allowing cell-cycle progression [8.Burke R.M. et al.Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3436-E3445Crossref PubMed Scopus (26) Google Scholar]. The SPRR2B-USP7 interaction is strongly induced by SPRR2B tyrosine-67 phosphorylation, presumably by the SRC nonreceptor tyrosine kinase [8.Burke R.M. et al.Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3436-E3445Crossref PubMed Scopus (26) Google Scholar]. Although direct involvement in MDM2–p53 signaling pathway has so far been reported only for SPRR2B, it is likely that, due to the high degree of homology and possession of the seemingly critical tyrosine-67 residue, other class members such as SPRR2A and SPRR2G can engage in analogous interactions. The engagement of SPRRPs in p53 signaling and in protection from DNA damage foreshadows their implication in tumorigenesis and in responses to DNA damage-based anticancer treatments. Indeed, SPRRPs are increasingly being linked to various human neoplasms. Of clinical interest are the findings indicating SPRRPs as plausible cancer biomarkers. Median serum SPRR2A concentration in gastric carcinoma (GC) patients was significantly higher than in healthy controls, exhibiting 75.7% sensitivity and 74.5% specificity [9.Xu X. et al.Serum small proline-rich protein 2A (SPRR2A) is a noninvasive biomarker in gastric cancer.Dis. Markers. 2020; 8493796Crossref Scopus (4) Google Scholar]. SPRR2A serum levels in GC patients were significantly associated with lymph-node metastasis and the tumor–node–metastasis (TNM) stage (P < 0.05). In a similar fashion, SPRR2A was one of the most consistently downregulated transcripts in metastatic and recurrent cells in a panel encompassing patient-matched primary and secondary head-and-neck squamous-cell carcinoma (HNSCC) models [10.Nisa L. et al.Comprehensive genomic profiling of patient-matched head and neck cancer cells: a preclinical pipeline for metastatic and recurrent disease.Mol. Cancer Res. 2018; 16: 1912-1926Crossref PubMed Scopus (19) Google Scholar]. Furthermore, SPRR2A was downregulated in HNSCC primary tumors (61.9% of cases) and lymph-node metastases (31.3%), but not in normal tissue. High expression of SPRR2A in lymph-node metastases was identified as an independent prognostic factor for regional disease recurrence after surgery and radiotherapy [10.Nisa L. et al.Comprehensive genomic profiling of patient-matched head and neck cancer cells: a preclinical pipeline for metastatic and recurrent disease.Mol. Cancer Res. 2018; 16: 1912-1926Crossref PubMed Scopus (19) Google Scholar]. These findings are likely to pave the road for exploration of additional features that relate SPRRPs to the onset, maintenance, and progression of malignant disorders. The first hint of a role for SPRRPs in antimicrobial defense was the observation that colonization of a germ-free mouse intestine with the commensal Bacteroides thetaiotaomicron markedly increases expression of mouse SPRR2A [11.Hu Z. et al.Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.Science. 2021; 374eabe6723Crossref Scopus (24) Google Scholar]. SPRR2A expression was triggered by exposure to the bacterial cell wall component lipopolysaccharide (LPS), mediated through TLR4-MyD88 signaling and mostly restricted to goblet and Paneth cells of the intestinal epithelium, from which SPRR2A was secreted in the intestinal lumen [11.Hu Z. et al.Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.Science. 2021; 374eabe6723Crossref Scopus (24) Google Scholar]. SPRR2A, which has under physiological conditions basic pH, subsequently interacted with the bacterial membrane by binding to lipids bearing negatively charged headgroups, thus disrupting bacterial membranes and eliciting a dose-dependent reduction in the viability of the Gram-positive species Listeria monocytogenes, Enterococcus faecalis, and Lactobacillus reuteri (Figure 1E). Interestingly, despite the ability of LPS to induce SPRR2A expression, Gram-negative bacteria were unaffected by this phenomenon as LPS neutralized SPRR2A-mediated membrane permeabilization [11.Hu Z. et al.Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.Science. 2021; 374eabe6723Crossref Scopus (24) Google Scholar]. SPRR2A also acts in antibacterial defense during helminth infections (e.g., by Heligmosomoides polygyrus), which can promote bacterial overgrowth and breach the intestinal barrier. This is mediated via type 2 cytokines interleukin 4 and interleukin 13, both secreted in response to helminth infection and stimulating SPRR2A expression. Compellingly, the helminth-induced protection against bacterial invasion makes SPRR2A unique among previously described antimicrobial proteins [11.Hu Z. et al.Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.Science. 2021; 374eabe6723Crossref Scopus (24) Google Scholar]. Analogous findings were reported for other SPRRP family members, which form an integral part of the cutaneous barrier in protection against skin infections [12.Zhang C. et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.Elife. 2022; 11e76729Crossref Scopus (12) Google Scholar]. Both LPS exposure of human immortalized sebaceous gland cells and LPS injection into the skin of germ-free mice resulted in a significant MYD88-mediated increase in mouse SPRR1A and SPRR2A expression (Figure 1E). Mouse as well as human SPRRPs displayed potent activity against a wide range of bacteria – including MRSA and P. aeruginosa (one of the most common causes of mortality in burn patients). SPRRPs bactericidal activity was equivalent to the previously described bacterial membrane disruption in intestinal bacteria [12.Zhang C. et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.Elife. 2022; 11e76729Crossref Scopus (12) Google Scholar], and these effects were replicated in vivo where Sprr1a–/– and Sprr2a–/– mice were more susceptible to MRSA and P. aeruginosa infections, indicating their integral role in the formation of a cutaneous barrier defending the host against systemic infection. Harmful processes such as inflammation and oxidative stress, both of which are known to increase SPRRPs expression, require rapid and efficient adaptation to maintain cellular survival. We highlight SPRRPs as essential components of the epithelial tissue defense machinery under such stressful conditions. SPRRPs quench ROS and directly protect cells from chromosomal damage, with consequent effects on genome stability through DNA binding. Complementarily, SPRRPs downregulate p53 expression by diminishing its acetylation and enhanced ubiquitination, thus making cell-cycle arrest and apoptosis less likely. This allows SPRRPs to act as active mediators in the cellular DNA damage response and implicates them in the pathophysiology of CHF, acute kidney injury, and cancer. Additionally, SPRRPs expression can alter cellular phenotype by inducing migratory capabilities and epithelial–mesenchymal transition, thus allowing rapid cell mobilization under physiological conditions, or affecting chemoradioresistance in pathological circumstances such as cancer. Furthermore, SPRRPs guard the host via bacterial membrane disruption, thereby providing additional protection against bacterial invasion. In the era of rapidly emerging multidrug-resistant bacterial strains and the worrying shortage of novel antibacterial therapies, these exciting new findings may offer next-generation avenues to tackle frequently lethal pathogens, such as MRSA and P. aeruginosa. A major challenge in the SPRRPs field is represented by the high homology between SPRRPs, which in some cases differ only in one amino acid. This presumably leads to a high degree of biological redundancy and the capability of stringent regulation through a tight differential tissue/cell-type expression. Consequently, various members of the family may carry out analogous functions in different tissues and fine-tune cellular response to different or specific stimuli (e.g., induction of SPRR2 family members in response to UV light in keratinocytes, induction of SPRR2B in response to ROS exposure in cardiac fibroblasts, or induction of SPRR2A in response to LPS in intestinal goblet cells). Conversely, other biological functions, such as DNA binding by SPRR4 or direct engagement in MDM2-p53 signaling by Y67-bearing SPRR2 proteins, appear to be restricted to one class or certain class members and dictated by minor but relevant alterations in protein primary structure. Further research is necessary to decipher these fine nuances and gain a complete understanding of this captivating family of proteins and their physiologic regulation. This work has been supported by a Swiss National to are of interest to a of proteins whereby an acetyl is onto a residue, affecting its a or characteristic by which a pathological or physiological process or disease can be an the membrane of epithelial cells and essential for providing protection against and the removal of acetyl groups from a under conditions between the groups of cysteine residues in a process by which epithelial cells their cell and and gain migratory and a of a of mostly of epithelial cell in the the of the component of the cell wall in the membrane of Gram-negative bacteria of a and a a critical of the tumor it a role in p53 transcriptional protein and a protein expression is induced by DNA it acts as a tumor by stimulating cell-cycle DNA cellular or or that have one or more which makes them a in which a ubiquitin is to a the protein for degradation via the degradation

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