SerpinB5, also known as maspin, is considered a tumor suppressor serpin that does not present itself as a protein inhibitor like others of its own family, the serine protease inhibitor superfamily (serpins). Maspin was first identified in 1994 on mammary tissue and breast cancer cell lines ^[1], but it is also known to be expressed on a wide range of cell types and tissues, mainly in epithelial cells, i. e. in prostate, lung, skin, and corneal stromal cells ^[2]. It differs from ordinary serpins once it does not undergo the stressed (S) to relaxed (R) conformation which is a striking feature of other proteins in serpin’s superfamily ^[1]. Instead, its G-helix has quite a flexibility, capable of changing the conformation of the protein itself ^[3].

Maspin and its superfamily

Serpins

Serpins usually inhibit other proteins like serine proteases, caspases and papain-like cysteine proteases, however, some of them do not accomplish an inhibitory role (3). As an example, some of them function as hormone transporters, molecular chaperones or even as tumor suppressors (3). Serpins structure usually contain three ß-sheets (A, B and C) and eight to nine 𝛂-helices (hA-hI) on their structure, and the most important region to interact with their targets is the reactive center loop (RCL) (An overview of the serpin superfamily). Inhibitory serpins are considered “suicide molecules” because they can only be used once (Huntington, J., Read, R. & Carrell, R. Structure of a serpin–protease complex shows inhibition by deformation . Nature 407, 923–926 (2000). https://doi.org/10.1038/35038119). The RCL is usually positioned out of the body of the serpins. When inhibiting proteases, serpins get their RCL cleaved out of the main structure, causing the amino-terminal portion of the RCL to form an additional fourth strand called s4A, once it is inserted into the center of ß-sheet A. This cleavage and modification on the structure of serpin is called the ‘stressed (S) to relaxed (R) transition’, in which the protein is in its biologically active state and transitions to a more thermal stable and latent state, respectively (An overview of the serpin superfamily).

Maspin

Maspin is a 42 kDa protein (Zou Z, Anisowicz A, Hendrix MJ, et al. Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 1994; 263: 526–9.) and is inserted in clade B of the serpin superfamily, composed of papain-like enzymes and inhibitory serpins that target cytotoxic apoptotic proteases which are working incorrectly (3). Differently from other serpins, Maspin does not undergo the S to R transition (An overview of the serpin superfamily). Instead, its G-helix is capable of undergoing a significant conformational change, that means this region of the molecule has some flexibility that allows movement. However, it is important to mention that studies have demonstrated, by superposing all of the maspin chains, a conformational heterogeneity at and around the G-helix (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). Also maspin is not limited to a certain cell compartment, once it is found on nucleus, cytoplasm, membrane, and as a secreted protein, according to the cell type and tissue (1)(2). Currently, it is known that the subcellular location of maspin is important for its tumor suppressor activity, and not only its protein levels inside the cell. In the past, there was a controversy about it, once maspin was upregulated in some tumors, while downregulated in others (Nuclear localization of maspin is essential for its inhibition of tumor growth and metastasis). Then, its translocation to the nucleus was observed and maspin’s nuclear localization was related to its tumor suppressor function, (Nuclear localization of maspin is essential for its inhibition of tumor growth and metastasis). However, contrary to what is expected, it has never been found a nuclear localization sequence (NLS), nuclear export sequence (NES), neither a secretory leader sequence (SLS) on maspin structure (Bodenstine TM, Seftor REB, Khalkhali-Ellis Z, Seftor EA, Pemberton PA, et al. (2012) Maspin: molecular mechanisms and therapeutic implications. Cancer and Metastasis Reviews 31: 529–551.). The tumor suppressor function of maspin is probably related to its activities, which are mainly inhibition of cell growth, invasion, tumoral migration, apoptosis stimuli, gene transcription regulation, angiogenesis inhibition (4) and prevention of oxidative damage of the proteome (5). Besides all of these functions, maspin also has an important role in the organization of the epiblast during early embryonic development (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). However, maspin lacks studies on non-tumoral cell lines, and its role on a normal condition might be different from its activity inside a tumoral lineages.

Relevance

Function and Structural highlights

RCL

Maspin structure does not differ a lot from other clade B serpins. It has three ß-sheets, nine 𝛂-helices and a reactive center loop (RCL). The latter is exposed in ordinary serpins and has a great flexibility. Serpins that have mutations within their RCL which interfere with the ability to undergo the stressed (S) to relaxed (R) conformational change cannot inhibit proteases and maspin’s RCL is the one among serpins that has the most different sequence (Al-Ayyoubi M, Gettins PGW, Volz K (2004) Crystal structure of human maspin, a serpin with antitumor properties - Reactive center loop of maspin is exposed but constrained. Journal of Biological Chemistry 279: 55540–55544.; Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, et al. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins - Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. Journal of Biological Chemistry 276: 33293–33296. Law RH, Irving JA, Buckle AM, Ruzyla K, Buzza M, et al. (2005) The high resolution crystal structure of the human tumor suppressor maspin reveals a novel conformational switch in the G-helix. Journal of Biological Chemistry 280: 22356–22364.).

Maspin does not present the conformational switch already discussed and does not have the consensus motif present in other serpins (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). The intact RCL is necessary for maspin’s activity as a tumor suppressor (Sheng, S., Pemberton, P. A., and Sager, R. (1994) J. Biol. Chem. 269, 30988 –30993), but there is no rearrangement of this structure, in other words, there is no S to R conformational change (Pemberton, P. A., Wong, D. T., Gibson, H. L., Kiefer, M. C., Fitzpatrick, P. A., Sager, R., and Barr, P. J. (1995) J. Biol. Chem. 270, 15832–15837 Bass, R., Moreno Ferna´ndez, A.-M. M., and Ellis, V. (2002) J. Biol. Chem. 277, 46845– 46848). Besides that, the RCL alone has been related to cell matrix adhesion and inhibition of cell invasion (Ngamkitidechakul, C., Warejcka, D. J., Burke, J. M., O’Brien, W. J., and Twining, S. S. (2003) J. Biol. Chem. 267, 31796 –31806).

One of the reasons for maspin’s RCL being unable to undergo the conformational switch is its limited flexibility, as it is not flexible like other serpins (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). The RCL of Maspin is shorter by four residues and lies closer to the serpin core of the molecule, it is positioned further “back”, in other words closer to the N-terminal, than all of the other known serpin RCL structures (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties). Besides that, the RCL of Maspin is stabilized by bonding interactions with amino acid side chains of the ß-sheet C, leading to a more rigid structure (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties). Additionally, the breach, where the cleaved RCL is inserted and which is present in other serpins, is not seen on maspin (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties).

A curious phenomenon that happens with maspin is the aggregation of dimers of tetramers, generating octamers in vitro. There is strong evidence that the hydrophobic residues on the RCL are responsible for the aggregation (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties). The hydrophobic residues Ile-344, Val-336, Ile-341, Leu-342, Pro-337 and adjacent aminoacids present on the RCL are completely exposed to the solvent (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties). Other types of intermolecular interactions, like minor salt links and hydrogen bonds between the s3C and s4C strands of opposing tetramers, also contribute to maintaining the structure, but the main force that results in the octamer are the hydrophobic associations (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties). Taking into account that the RCL is responsible for functions of cell matrix adhesion and inhibition of cell invasion (Ngamkitidechakul, C., Warejcka, D. J., Burke, J. M., O’Brien, W. J., and Twining, S. S. (2003) J. Biol. Chem. 267, 31796 –31806), its hydrophobic nature is expected to be functionally important (Crystal Structure of Human Maspin, a Serpin with Antitumor Properties).

Further research is needed to understand if this phenomenon also happens in vivo conditions. The RCL might be important for defining the protein subcellular localization. Modification of the Aspartate 346 (D346) by a glutamic acid (E) residue on the C-terminal portion of RCL in maspin leaded maspin to a dominant nuclear distribution and increased interaction with HDAC1 in multiple cancer cell lines (Identification of an intrinsic determinant critical for maspin subcellular localization and function).

Bulge around D and E-helices

Maspin also contains a buried salt bridge on the periphery of the conserved region called ‘shutter’ which is located in the center of the serpin fold that is important for controlling conformational change in inhibitory molecules. This region, which is formed by residues from D-helix, B-helix and s2A, causes a prominent bulge at the N-terminal end of s1A and reveals a cavity beneath the D-helix (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). It suggests that the salt bridge region, as in other serpins, may have some relevance at the interaction with a binding partner. Furthermore, the distortion in secondary structure caused by the salt bridge introduces Lys114 into the center of a cluster of conserved positively charged waste. Once maspin is able to bind heparin, it is possible that these residues perform as a heparin binding site (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix).

G-helix

Maspin is able to undergo conformational change in and around the G-helix, and has an open and closed form. This is a real putative cofactor binding site and may determine maspin’s function (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix). As said before, the region at and around the G-helix has flexibility to undergo a conformational change and, as a consequence, its charged residues are reorganized on the G-helix and a central part of the helix structure becomes negative. The two structures, reflecting maspin in the open and closed conformation, show that rotation of the G-helix alters the local charge distribution, suggesting that this movement represents a conformational “switch”, what researchers have implied be a cofactor binding site under conformational control based on G-helix negative charged patch modulation (The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix).

Physical interactions

It has been known that maspin interacts physically with a lot of different proteins. We will specifically discuss two of them below.

Histone Deacetylase 1 (HDAC1)

Maspin is able to bind to histone deacetylase 1 (HDAC1), which is up-regulated in many types of cancers and is an important class I nuclear deacetylase (Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function). Either purified or endogenously expressed maspin is bound to and inhibits HDAC1 (Li X, Yin S, Meng Y, Sakr W, Sheng S (2006) Endogenous inhibition of histone deacetylase 1 by tumor-suppressive maspin. Cancer Res 66: 9323–9329.). This interaction and, consequently, the inhibition of HDAC1, may allow maspin to control a small set of genes involved in epithelial differentiation (Bernardo MM, Meng Y, Lockett J, Dyson G, Dombkowski A, et al. (2011) Maspin reprograms the gene expression profile of prostate carcinoma cells for differentiation. Genes Cancer 2: 1009-1022.). The inhibition of HDAC1 by maspin lead to increased acetylation of HDAC1 target protein Ku70, which in turn, caused an increase in apoptosis (Lee SJ, Jang H, Park C (2012) Maspin increases Ku70 acetylation and Bax-mediated cell death in cancer. Int J Mol Med 29: 225–230.).

Unfortunately, it is not known where the exact binding site of maspin to HDAC1 is until the date of creation of this page (07/20/2022) and more studies are still needed.

Glutathione S-Transferase (GST)

The Glutathione S-Transferase (GST) is an important molecule for the regulation of ROS-induced signaling and accomplishes an antioxidant role (Antioxidant Role of Glutathione S-Transferases: 4- Hydroxynonenal, a Key Molecule in Stress-Mediated Signaling). It is known that maspin interacts physically with GST, thus regulating the cell's response to oxidative stress (Yin S, Li X, Meng Y, Finley RL Jr, Sakr W, Yang H, Reddy N and Sheng S: Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione Stransferase. J Biol Chem 280: 34985-34996, 2005). Besides that, a new oxidized modification on maspin has been found, which has a different isoelectric point (Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress). The level of oxidized maspin increased in accordance with oxidative stress. Surprisingly, the authors found that the oxidized form of maspin had lower binding affinity to GST. They concluded that “the intramolecular disulfide-bonded formation of maspin might have some distinct properties compared to the native maspin isoforms under oxidative stress” (Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress). Even though the authors couldn’t define exactly the molecular function of this new modification on maspin, they presumed it might affect biochemical properties of the protein and its subcellular localization. Another hypothesis is that the modification might protect maspin from oxidative stress damages, which may result in an increase in the half-life of the protein (Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress). Impressively, modification of the residue R340 of maspin RCL, to an Alanine, causes the protein to lose affinity with GST and leaded to a lower GST activity (Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase). The authors imagine that maspin might play a regulatory role instead of acting directly as a chaperone or as a detoxifying enzyme (Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase). Until the present moment, it has not been described the exact interaction site between GST and maspin, but the authors hypothesize an important role for the RCL on this interaction (Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase).

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