Dnases in health and disease 

Author: Peter A. Keyel*

ABSTRACT

DNA degradation is critical to healthy organism development and survival. Two nuclease families that play key roles in development and in disease are the Dnase1 and Dnase2 families. While these two families were initially characterized by biochemical function, it is now clear that multiple enzymes in each family perform similar, non-redundant roles in many different tissues. Most Dnase1 and Dnase2 family members are poorly characterized, yet their elimination can lead to a wide range of diseases, including lethal anemia, parakeratosis, cataracts and systemic lupus erythematosus. Therefore, understanding these enzyme families represents a critical field of emerging research. This review explores what is currently known about Dnase1 and Dnase2 family members, highlighting important questions about the structure and function of family members, and how their absence translates to disease.

DNA的降解对于机体的健康发育以及生存非常重要。在机体发育过程中发挥重要作用的两种核酸酶家族分别是Dnase1以及Dnase2。这两种家族的生物化学功能已经被阐述的很清楚，每个家族中的核酸酶都会在很多组织中发挥相似的、非冗余的作用。很多Dnase1以及Dnase2家族成员都没有被完全阐述，然而他们的缺失会导致非常多疾病，包括致命性贫血，角化不全，白内障和系统性红斑狼疮。因此，理解这些核酸酶家族对于生物机体研究非常重要。这里将综述目前所知的Dnase1以及2的家族成员，重点放在他们的结构和功能上，以及它们的缺失所导致的疾病。

1. Introduction

Regular development and healthy function of humans, mice and other organisms requires two understudied families of endonucleases that specifically target DNA called Dnases. These two Dnase families were initially assumed to be only two enzymes, based on the biochemical properties of their respective DNase activities. Dnase1 activity typically requires divalent cations (Ca2+ or Mg2+), shows peak activity at neutral pH, and leaves 5’ phosphates following DNA cleavage (Table 1)(Shiokawa and Tanuma, 2001). In contrast, Dnase2 activity typically does not require divalent cations, shows peak activity at an acidic pH, and leaves 3’ phosphates following DNA cleavage (Table 1) (Counis and Torriglia, 2006; Evans and Aguilera, 2003). 

无论是人体还是小鼠或者其他的机体，都需要两种家族的专门针对DNA的核酸酶来保证机体健康，那就是Dnase。这两个家族最开始只是用来指代两个酶，是根据它们各自的理化特性。Dnase1的活性基本都需要二价阳离子（比如Ca2+，Mg2+），在中性pH时活性最高，并且在DNA切割之后留下5'的磷酸。相反地，Dnase2的活性基本不需要二价阳离子，在酸性条件下活性最好，切割DNA后留下3'磷酸。

However, through molecular cloning it became clear that these DNase activities are shared by multiple, related enzymes. While most of these enzymes are full-time nucleases, one Dnase2 enzyme arises from the transformation of the serine protease inhibitor SerpinB1 (also called Monocyte/Neutrophil Elastase Inhibitor/MNEI or Leukocyte Elastase Inhibitor/LEI) into L-Dnase II (Padron-Barthe et al., 2007; Torriglia et al., 1998). These Dnases have varied historical names, but are now methodically named Dnase1, Dnase1L1, Dnase1L2, Dnase1L3, Dnase2a, Dnase2b, L-DnaseII (Table 1). The diversity of these enzymes allow the body to regulate Dnase activity in different organs according to the needs of that organ. 

然而，通过分子克隆我们逐渐明白这些Dnase的活性很多酶都具有。虽然绝大多数酶完全（full-time）行使核酸酶的功能，但有一种Dnase2从丝氨酸蛋白酶抑制剂（serine protease inhibitor）SerpinB1（也被称为单核细胞/中性粒细胞弹性蛋白酶抑制剂，即MNEI，或者白细胞弹性蛋白酶抑制剂，即LEI）向L-Dnase II转化过程中产生的。这些Dnase有很多曾经的名字，但现在都系统命名为Dnase1, Dnase1L1, Dnase1L2, Dnase1L3（Dnase1家族）, Dnase2a, Dnase2b, L-DnaseII（Dnase2家族）。这些酶的多元性使得机体可以在不同器官中调节Dnase活性来适应不同器官的需求。

Overall, the absence of members from these two families of Dnases leads to a wide variety of diseases (Table 2), highlighting the necessity of regulated degradation of endogenous DNA throughout development. This review will examine the tissue expression of Dnase1 and Dnase2 families, their structural and functional characteristics, their roles in disease and their mechanisms of action. Based on this current knowledge, this review will also consider important needs and questions in this maturing field.

总之，Dnase家族成员的缺失会导致非常多疾病，因为机体发育过程中内源DNA的调节性降解的必要性。本综述Dnase1以及2的结构与功能，以及它们的各种疾病中的机制。

3. Structure of Dnases

The structure of Dnase1 and Dnase2 family members can help illuminate the unique functions of each nuclease. Based on primary sequence and predicted/known structures, the Dnase1 and Dnase2 families fit within the Dnase superfamily of proteins as two distinct groups. Dnase1 and Dnase2 families show low sequence homology to each other, but within each family, the nucleases are strongly conserved. The Dnase1 family shows 24% identity and 51% homology between all four family members (Fig. 1A). Likewise, Dnase2a and Dnase2b are also strongly conserved, with 35% identity and 66% homology between them (Fig. 1B). In contrast, L-DnaseII is poorly conserved with other Dnase2 family members. Overall, L-DnaseII shows only 29% homology with the other two Dnase2 family members, which is consistent with its alternate activity as SerpinB1.

了解Dnase的结构可以帮助阐述它们之间不同的功能。基于基本序列以及已经被测明的结构，Dnase的两个家族有非常大的区别，两个家族之间的同源序列非常少，但是在同一家族中却非常保守。Dnase1家族有24%的一致性以及51%的同源性。而Dnase2a以及2b非常保守，它们之间有35%的一致性与66%的同源性。而L-Dnase ll与其他Dnase2家族的成员结构非常不保守，L-Dnase ll只与它们有29%的同源性，这也侧面证实它可以替代SerpinB1的功能。

 Despite the weak interfamily conservation in the primary sequence, both Dnase families share a similar, simple domain architecture. The major feature in these proteins is a Dnase domain, which spans the majority of the protein (Fig. 2). The Dnase domain forms the basis for the Dnase superfamily of enzymes, so named from the crystallization of Dnase1 (Suck et al., 1984). The Dnase superfamily of enzymes generally acts as phosphohydrolases. Superfamily members exclude CAD (Woo et al., 2004), but include other endonucleases like apurinic/apyrimidinic DNA repair enzymes (Dlakic, 2000; Weichenrieder et al., 2004), lipid phosphohydrolases like bacterial and neutral sphingomyelinases (Matsuo et al., 1996; Clarke et al., 2006) and inositol polyphosphate5-phosphatases like synaptojanin (Dlakic, 2000; Whisstock et al., 2000; Tsujishita et al., 2001). Dnase1 and Dnase2 proteins fit well into this superfamily, since they cleave the DNA phosphodiester bond, albeit on different sides of the phosphate. Dnase1 and Dnase2 families bear other family-specific differences discussed below.

尽管基本序列上不同家族之间的同源性非常低，但是两个家族都有非常简单和相似的结构域。它们最主要的结构特征就是有一个DNase结构域，在整个蛋白中范围最广。Dnase结构域构成了Dnase家族酶的基础。所有Dnase基本上都作为磷酸水解酶……Dnase1与Dnase2蛋白在这个超级家族中非常体面，因为它们都可以切割DNA的磷酸双酯键，尽管是从磷酸键的不同侧面去切割。Dnase1与Dnase2其它的差异将会在下文介绍。

3.1. Dnase1 family

All Dnase1 family members contain a signal sequence prior to the Dnase domain which directs protein insertion into the endoplasmic reticulum (ER). The signal sequence is typically 18–22 amino acids long, but shows very little homology between family members (Fig. 1A). Functionally, the signal sequence can inhibit Dnase activity (Shiokawa et al., 1998; MacLea et al., 2003) along with its typical function of promoting ER translocation (Shiokawa and Tanuma, 2001; Shiokawa et al., 1998; Shak et al., 1990). After cleavage of the signal sequence in Dnase1 and Dnase1L3, both are primarily secreted (Sisirak et al., 2016; Napirei et al., 2005; Nishikawa et al., 1997). In contrast, following cleavage Dnase1L1 remains anchored to the membrane through a glycosylphosphatidylinositol (GPI) anchor and is localized to the ER and plasma membrane (Shiokawa et al., 2007). Based on unpublished overexpression data, Dnase1L2 might reside in the ER (Fischer et al., 2011), though no ER retention motif has been characterized, and it could instead traffic to/ reside in the lysosome similar to other Dnase1 and Dnase2 proteins (Nishikawa et al., 1997; Sleat et al., 2006; Nakahara et al., 2007). Consequently, all Dnase1 family members are thought to act either in the lumen of organelles or extracellularly. However, intracellular roles have been described for two Dnase1 family members, Dnase1L2 and Dnase1L3. 

所有Dnase1家族成员都在Dnase结构域前面有一个信号序列段，它可以引导蛋白质插入ER。信号序列基本上都是18-22个氨基酸，但是在家族成员之间的同源性非常低。功能上来讲，信号序列可以抑制Dnase的活性，并且可以促进ER重定位。在对Dnase1与Dnase1L3的信号序列进行切割之后，它们主要是被分泌（而不是在内质网上）。相反，切割后的Dnase1L1通过糖基磷脂酰肌醇(GPI)锚定在膜上，并定位于内质网和质膜上(Shiokawa等，2007)。根据未发表的过表达数据，Dnase1L2可能驻留在内质网(Fischer等，2011)，尽管没有内质网保留基序的特征，但它可能会运输到/驻留在溶酶体中，类似于其他Dnase1和Dnase2蛋白(Nishikawa等，1997;Sleat等人，2006;Nakahara等人，2007)。因此，所有的Dnase1家族成员被认为在细胞器腔内或细胞外活动。然而，已有两个Dnase1家族成员dnnase1l2和Dnase1L3在细胞内的作用被描述。