See Article by J. Li et al

Defects in protein homeostasis (also known as proteostasis) are intrinsically linked to age-related decline of cardiac function and likely contribute to cardiomyopathies. Protein-folding chaperones and protein quality control systems work overtime in the high stress cardiac environment, responding to wear and tear. Tight regulation of the pathway components is often controlled by posttranslational modifications. In addition to compact posttranslational modifications such as phosphorylation, bulkier posttranslational modifications involve Ub (ubiquitin) and other small UbL (Ub-like) proteins, which are covalently conjugated to target proteins. These modifications can affect stability, localization, and function of the target protein. Ufm1 (ubiquitin-fold modifier 1) is a less-studied UbL, but modification by Ufm1 (ufmylation¹) is emerging as an important mediator of the endoplasmic reticulum (ER) stress response, which is activated in cardiomyocytes during heart failure. Focusing on Ufl1 (Ufm1-ligase 1), one of the enzymes that mediate ufmylation, the report from Li et al² provide strong evidence that Ufl1 has a cardioprotective role and points to the ufmylation pathway as a potential target for pharmacological intervention for some cardiomyopathies.

Ub and UbLs share a common biology in the way they are added to targets and recycled in the cell, but each class tends to have specialized roles.³ They are varied in their individual protein sequences, with Ub/UbLs sharing a common structural fold as revealed by crystallographic as revealed by protein crystallographic studies. After synthesis as a pro-protein, Ub/UbLs are cleaved at the C terminus by a protease, a prerequisite for entering the conjugation cycle. The cleaved Ub/UbL is engaged by an activation enzyme (generically termed E1 or enzyme 1). Via this Ub-E1 intermediate, the Ub/UbL is then passed to a conjugating enzyme (E2 or enzyme 2). In some cases, the E2 can engage directly with target proteins, and the Ub/UbL is conjugated by its C terminus to the targets, usually to a lysine. In most cases, however, the Ub-E2 intermediate engages with a ligating enzyme (or E3 ligase; enzyme 3). The structure of the E3 may vary greatly, from being a single protein to a multiprotein complex, but in general, the E3 serves to enhance delivery of the mature Ub/UbL to a lysine on specific target proteins. Certain conserved protein domains are characteristic of E3 ligases (eg, RING [really interesting new gene] or HECT [homologous to the E6-AP carboxyl terminus] domains), and by this measure, it is estimated that >600 E3 Ub/UbL ligases are encoded by the human genome. Other E3 ligases have been described that lack these domains but may function as scaffolding adaptors to bridge the E2-Ub/UbL and targets (Ufl1, featured in Li et al,² is one example). Ub/UbL posttranslational modifications vastly increase the complexity of the human proteome and require exquisite regulation to allow cells to cycle, survive, and respond to signals and stress.

Correct regulation of proteostasis underlies both healthy and diseased/damaged heart function. This complex task balances protein synthesis, folding, and degradation, with the latter relying on multiple systems (autophagy, calpains, proteasomes^4,5). Specifically, contributions to cardiac proteostasis by the Ub-proteasome system have been extensively reviewed.^6,7 Cardiac roles for modifications by other UbLs (NEDD8 [neural precursor cell expressed, developmentally down-regulated 8], SUMO [small ubiquitin modifier]) have also been described.^8,9 Many of the studies defining cardiac roles for Ub/UbL modifications rely on either classical or conditional knockout mice, with the latter allowing heart-specific deletion of the genes encoding the UbL or its associated pathway enzymes. These precise genetic studies allow molecular, histological, and physiological analysis of the heart in experimental animals. When coupled with surgical methods to induce heart stress or with drug administration or both, such studies can reveal promising targets and therapies for future clinical trials.

Ufmylation was discovered by chance in a search for proteins implicated in autophagy,¹⁰ and like ubiquitylation, it occurs through a multienzyme cascade (Figure). The E1-activating enzyme Uba5 (ubiquitin like modifier activating enzyme 5) was identified first, and further biochemical and proteomic analysis revealed Ufm1 as the novel UbL that is activated by Uba5. The same study also discovered the dedicated E2 enzyme, Ufc1 (UFM1 conjugating enzyme 1), responsible for conjugation of Ufm1 to target proteins. Later studies uncovered Ufl1, a Ufm1 E3 ligase, as an interactor of both Ufm1 and the E2 Ufc1.¹¹ The search for proteases that were capable of processing pro-Ufm1 and that could remove and recycle Ufm1 from target proteins yielded 2 related proteins, Ufsp1 and Ufsp2 (UFM1 specific peptidase 1 and 2).¹² All of these proteins show close association to the ER, and functional studies (including the present report) point to specialized roles for ufmylation in regulating ER-related stress. The ER is a major site of protein quality control, housing the synthesis machinery for proteins destined to be secreted or membrane-trafficked within the cell, as well as chaperones to assist protein folding and 3 separate systems (PERK [protein kinase R-like ER kinase], ATF6 [activating transcription factor 6], IRE1 [inositol-requiring enzyme 1]) that mediate the unfolded protein response. Several ufmylated target proteins have been identified, although these are few in number compared with those modified by Ub or SUMO. One ufmylated target, Ufbp1 (also known as Ddrgk1 [DDRGK domain containing 1]), warrants particular mention since it is required for efficient ufmylation of other targets and may act as a cofactor for the Ufl1 E3 ligase. Loss-of-function classical knockouts in mice (Uba5, Ufl1, or Ufbp1) have revealed an essential role for ufmylation in erythroid development and lead to embryonic lethality.¹ Tissue-specific knockouts can be used to reveal roles later in development, exemplified by Li et al.² This approach has uncovered a novel role for Ufl1 in mouse cardiac health.

Figure. Features of the ufmylation cycle, a key pathway for endoplasmic reticulum (ER)-based stress response in cardiac development and disease. UFM1 (ubiquitin-fold modifier 1) is conjugated to substrates through a multienzyme cascade. After proteolytic maturation, UFM1 is passed via 3 enzymes (activating E1, conjugating E2, and ligating E3) as it attaches to substrates. Specific proteases remove and recycle Ufm1. Li et al² demonstrate that Ufl1 (Ufm1-ligase 1), a Ufm1 E3 ligase, is cardioprotective in both physiological and pathological conditions. How ufmylation influences substrates, whether E3 ligases are obligatory, whether additional E3 ligases exist, and how the Ufbp2 cofactor affects E3 activity are still open questions for future investigation. Even so, the ufmylation pathway may offer unique drug targets for cardiac and other diseases that feature ER stress response as cause or consequence of the pathology. ATP indicates adenosine tri-phosphate; Ufbp1, UFM1-specific binding protein 1; and Ufc1, UFM1 conjugating enzyme 1.

Their study begins with the observation that, although ufmylation of major target proteins in the heart seems to increase over time (from 1-day to 1-year old), levels of Ufl1 E3 ligase seem to decrease. This may seem counterintuitive, but possible explanations may lie in altered levels or activities of other ufmylation mediators (Uba5, Ufc1, Ufbp1, Ufsp1/2). Upon surgically induced cardiac hypertrophy in mice, levels of both ufmylation and Ufl1 increased. Levels of Ufl1 decreased during induced ischemia/reperfusion, likely because of cardiomyocyte death. Samples from patients with dilated cardiomyopathy also showed reduced Ufl1 levels, all leading to the hypothesis that ufmylation might be a key player in cardiac stress response. To address this, the authors used a conditional mouse mutant to remove Ufl1 from the cardiomyocyte population, to see how this affected both normal heart development, as well as response to heart stress and injury.

While Ufl1 levels were significantly reduced in whole heart extracts from knockouts, detectable Ufl1 and ufmylation still remained, suggesting that genetic removal was not complete, or that noncardiomyocyte cells contribute to residual activity. Alternatively, as seen with SUMOylation, perhaps ufmylation of some targets does not require an E3 ligase, or perhaps another uncharacterized Ufm1 E3 ligase acts redundantly to Ufl1 (Figure). Still, reduction of Ufl1 was sufficient to induce cardiac remodeling and progressive functional deterioration as measured by echography, histology, and changes in gene expression. Using induced cardiac hypertrophy, Ufl1^CKO mice were unable to make compensatory changes and developed heart failure, with increased fibrosis and decreased contractility, suggesting that Ufl1 has a cardioprotective role.

Turning to molecular mechanisms, comparative analysis of transcriptomes from control and Ufl1^CKO mice pointed to ER dysfunction when Ufl1 was reduced. Indeed, as reported previously for cells and other organ systems, loss of Ufl1 and ufmylation in the heart leads to increased ER stress, evident as an increase in stress response chaperones and changes in ER ultrastructure. Induced cardiac hypertrophy also induced stress chaperones, but the same treatment in Ufl1^CKO mice led to even higher levels. Using primary cardiomyocytes from rat and drugs to induce ER stress, the authors show that at least 1 of 3 unfolded protein response pathways (PERK) was affected by Ufl1 reduction. Using a previously reported strategy,¹³ administration of tauroursodeoxycholic acid, a chemical chaperone known to alleviate ER stress, was able to reduce the cardiac enlargement and ER stress-induced cell death seen in Ufl1^CKO mice with surgically induced cardiac hypertrophy. Overall, the results convincingly show that Ufl1 and ER stress regulation are tightly connected and that ufmylation may be a druggable cardioprotective pathway.

Although tauroursodeoxycholic acid is being testing in clinical trials, including some focused on cardiac issues (eg, https://www.clinicaltrials.gov. Unique identifier: NCT01855360), a treatment more focused on Ufl1 and the ufmylation pathway may yield less secondary effects. Given the current push to exploit E3 ligases and deubiquitinases as druggable targets,^14,15 the ufmylation pathway will certainly be explored and new drug candidates will likely emerge in the upcoming years. Li et al² suggests that activation of ufmylation would be beneficial in the case of some cardiomyopathies and perhaps age-related decline of cardiac health. A better understanding of how Ufl1 interacts with targets for ufmylation and how Ufsp1 (the potential Ufl1 cofactor; Figure) contributes to the E3 activity of Ufl1 may lead to new ideas on how to find or design activating compounds. Activators of Uba5 and Ufc1 could also lead to overall increases of ufmylation. Conversely, a search for inhibitors of Ufm1 proteases may identify a compound capable of enriching for ufmylated proteins. A recent report shows that the loss of Ufsp2 in cells leads to a dramatic increase in ufmylated targets,¹⁶ suggesting that inhibitors may yield the desired effect. Since heart failure is a leading cause of morbidity and mortality, especially among middle-aged and older adults, this important study reveals ufmylation as a promising pathway to explore for therapeutic benefits in cardiomyopathies and likely other ER stress-based illnesses.

Drs Sutherland and Barrio are funded by BFU2017-84653-P (MINECO/FEDER, EU), the Severo Ochoa Excellence Program (SEV-2016- 0644), the UbiCODE program (765445, EU) and PROTEOSTASIS (COST BM1307, EU). Additional support was provided by the Department of Industry, Tourism, and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek Research Programs) and by the Innovation Technology Department of the Bizkaia County.

None.

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.

https://www.ahajournals.org/journal/circheartfailure

参见J. Li等的文章

蛋白质稳态（也称为蛋白稳态）的缺陷与年龄相关的心脏功能下降有内在联系，并可能导致心肌病。蛋白质折叠分子伴侣和蛋白质质量控​​制系统在高应力心脏环境中会超时工作，对磨损产生响应。途径组分的严格调节通常通过翻译后修饰来控制。除了紧凑的翻译后修饰（例如磷酸化）外，更大的翻译后修饰还涉及Ub（泛素）和其他小的UbL（Ub样）蛋白，它们与目标蛋白共价结合。这些修饰可影响靶蛋白的稳定性，定位和功能。Ufm1（泛素折叠修饰子1）是研究较少的UbL，但由Ufm1（泛素化）修饰¹）逐渐成为内质网（ER）应激反应的重要介体，后者在心力衰竭期间在心肌细胞中被激活。Li等[ ^2]的报告着眼于Ufl1（Ufm1-ligase 1），它是介导脲基化的酶之一，提供了有力的证据，证明Ufl1具有心脏保护作用，并指出ufmylation途径是某些心肌病的药理干预的潜在靶标。 。

Ub和UbLs被添加到靶标并在细胞中循环利用的方式具有共同的生物学特性，但每个类别往往都有专门的作用。³它们在各自的蛋白质序列中各不相同，Ub / UbLs具有共同的结构折叠，如通过晶体学揭示的（通过蛋白质晶体学研究揭示的）。合成为原蛋白后，Ub / UbLs在C末端被蛋白酶裂解，这是进入偶联循环的前提条件。裂解的Ub / UbL与激活酶（通常称为E1或酶1）结合。然后，通过该Ub-E1中间体，将Ub / UbL传递至结合酶​​（E2或酶2）。在某些情况下，E2可以直接与靶蛋白结合，并且Ub / UbL的C末端与靶标结合，通常与赖氨酸结合。但是，在大多数情况下，Ub-E2中间体与连接酶（或E3连接酶；酶3）结合。E3的结构可能会发生很大变化，从单一蛋白到多蛋白复合物，但一般来说，E3可以增强成熟Ub / UbL在特定靶蛋白上向赖氨酸的递送。某些保守的蛋白质结构域是E3连接酶的特征（例如RING [非常有趣的新基因]或HECT [与E6-AP羧基末端同源]结构域），通过这种方法，估计> 600 E3 Ub / UbL连接酶由人类基因组编码。已描述了其他E3连接酶，这些酶缺乏这些结构域，但可能充当支架衔接子，以桥接E2-Ub / UbL和靶标（Ufl1，在Li等人的文章中，据估计，人类基因组编码了＞ 600个E3 Ub / UbL连接酶。已经描述了其他E3连接酶，这些酶缺乏这些结构域，但可以充当支架衔接子，以桥接E2-Ub / UbL和靶标（Ufl1，在Li等人的文章中，据估计，人类基因组编码了＞ 600个E3 Ub / UbL连接酶。已经描述了其他E3连接酶，这些酶缺乏这些结构域，但可以充当支架衔接子，以桥接E2-Ub / UbL和靶标（Ufl1，在Li等人的文章中，²是一个示例）。Ub / UbL翻译后修饰极大地增加了人类蛋白质组的复杂性，并需要精确的调节以允许细胞循环，存活并响应信号和应激。

正确调节蛋白质变形是健康和患病/受损心脏功能的基础。这个复杂的任务平衡了蛋白质的合成，折叠和降解，而后者则依赖于多个系统（自噬，钙蛋白酶，蛋白酶体^4,5）。具体地，已经广泛地综述了Ub-蛋白酶体系统对心脏蛋白稳态的贡献。^6,7对其他UbL修饰的心脏作用（NEDD8 [表达的神经前体细胞，发育性下调8]，SUMO [小泛素修饰剂]）也已有描述。^8,9定义Ub / UbL修饰的心脏作用的许多研究都依赖于经典或条件敲除小鼠，后者允许心脏特异性删除编码UbL或其相关途径酶的基因。这些精确的遗传研究可以对实验动物的心脏进行分子，组织学和生理学分析。当与引起心脏压力的外科手术方法或与药物管理或两者一起使用时，此类研究可以揭示有希望的靶标和疗法，用于未来的临床试验。

泛素化是偶然发现的，涉及到与自噬相关的蛋白质[ ^10]，与泛素化一样，它是通过多酶级联反应发生的（图）。首先鉴定出E1激活酶Uba5（类似于泛素的修饰物激活酶5），进一步的生化和蛋白质组学分析表明，Ufm1是被Uba5激活的新型UbL。这项研究还发现了专门的E2酶Ufc1（UFM1偶联酶1），它负责将Ufm1偶联至目标蛋白。后来的研究发现，Ufl1是一种Ufm1 E3连接酶，可作为Ufm1和E2 Ufc1的相互作用体。¹¹对能够加工pro-Ufm1并能从目标蛋白中去除和回收Ufm1的蛋白酶的搜索产生了2个相关蛋白，Ufsp1和Ufsp2（UFM1特异性肽酶1和2）。¹²所有这些蛋白质均显示出与ER密切相关，功能研究（包括本报告）指出，磺酰化在调节ER相关应激中的特殊作用。ER是蛋白质质量控​​制的主要场所，拥有旨在在细胞内分泌或膜运输的蛋白质的合成机制，以及辅助蛋白质折叠的分子伴侣和3个独立的系统（PERK [蛋白激酶R样ER激酶]，ATF6 [激活转录因子6]，IRE1 [需要肌醇的酶1]）介导未折叠的蛋白质反应。已经鉴定了几种磺酰化的靶蛋白，尽管与被Ub或SUMO修饰的蛋白相比，其数量很少。一个乌米酰化的靶标，Ufbp1（也称为Ddrgk1 [DDRGK域包含1]），值得特别提及，因为它是有效靶向其他靶标的必需品，并可能充当Ufl1 E3连接酶的辅助因子。小鼠（Uba5，Ufl1或Ufbp1）功能丧失的经典基因敲除已显示出在肌红素发育中磺酰化的重要作用并导致胚胎致死率。¹组织特异的基因敲除可用于揭示后来在发育中的作用，例如Li等人的研究。²这种方法发现了Ufl1在小鼠心脏健康中的新作用。

数字。 ufmylation周期的特征，这是心脏发育和疾病中基于内质网（ER）的应激反应的关键途径。UFM1（泛素折叠修饰剂1）通过多酶级联反应与底物缀合。在蛋白水解成熟后，UFM1通过3种酶（激活E1，结合E2和连接E3）通过，附着在底物上。特定的蛋白酶可去除并回收Ufm1。李等²证明Ufl1（Ufm1-连接酶1），一种Ufm1 E3连接酶，在生理和病理条件下均具有心脏保护作用。富马酰化如何影响底物，是否必须使用E3连接酶，是否存在其他E3连接酶以及Ufbp2辅助因子如何影响E3活性仍是未来研究的问题。即便如此，磺酰化途径仍可为心脏病和其他以ER应激反应为病理原因或结果的疾病提供独特的药物靶标。ATP表示三磷酸腺苷；Ufbp1，UFM1特异性结合蛋白1; 和Ufc1，UFM1结合酶1。

他们的研究从观察开始，尽管心脏中主要靶蛋白的磺酰化似乎随时间（从1天到1岁）增加，但Ufl1 E3连接酶的水平似乎降低了。这似乎是违反直觉的，但可能的解释可能在于其他乌磺酰化介质（Uba5，Ufc1，Ufbp1，Ufsp1 / 2）的水平或活性改变。在小鼠手术诱发的心脏肥大后，磺酰化和Ufl1的水平均增加。在诱导的缺血/再灌注过程中，Ufl1的水平下降，可能是由于心肌细胞死亡所致。扩张型心肌病患者的样本也显示Ufl1水平降低，所有这些都提出了假设，磺酰化可能是心脏应激反应的关键因素。为了解决这个问题，作者使用条件小鼠突变体从心肌细胞群中去除了Ufl1，

虽然敲除的全心提取物中的Ufl1水平显着降低，但仍存在可检测的Ufl1和ufmylation，表明遗传去除不完全，或者非心肌细胞有助于残留活性。另外，从SUMOylation中可以看出，某些靶标的泛酰化作用可能不需要E3连接酶，或者另一个未表征的Ufm1 E3连接酶对Ufl1会产生多余的作用（图）。尽管如此，通过超声检查，组织学和基因表达的变化测量，Ufl1的减少足以诱导心脏重塑和进行性功能恶化。使用诱发的心肌肥大，Ufl1 ^CKO 小鼠无法进行代偿性变化并发展为心力衰竭，并伴有纤维化增加和收缩力下降，提示Ufl1具有心脏保护作用。

关于分子机制，对照和Ufl1 ^CKO小鼠的转录组的比较分析指出，当Ufl1减少时，ER功能障碍。确实，如先前关于细胞和其他器官系统的报道，心脏中Ufl1的丢失和磺酰化会导致内质网应激的增加，这表现为应激反应分子伴侣的增加和内质网超微结构的变化。诱导的心脏肥大也诱导了应激伴侣，但在Ufl1 ^CKO小鼠中进行相同的处理导致更高的水平。作者利用大鼠和药物中的原代心肌细胞诱导内质网应激，发现3个未折叠的蛋白质反应途径（PERK）中至少有1个受Ufl1降低的影响。使用先前报告的策略，¹³牛磺去氧胆酸（一种已知可减轻ER应激的化学伴侣）的给药，能够减少因外科手术诱发的心脏肥大的Ufl1 ^CKO小鼠的心脏增大和ER应激诱导的细胞死亡。总体而言，结果令人信服地表明，Ufl1和内质网应激调节紧密相关，而磺酰化可能是可保护性的心脏保护途径。

尽管牛磺去氧胆酸正在临床试验中进行测试，包括一些针对心脏问题的试验（例如，https：//www.clinicaltrials.gov。唯一标识符：NCT01855360），但更侧重于Ufl1和ufmylation途径的治疗可能产生较少的次要作用。鉴于当前将E3连接酶和去泛素酶开发为可药物化靶标的努力，[ ^14,15]肯定会探索磺酰化途径，并且在未来几年中可能会出现新的候选药物。李等²提示在某些心肌病以及可能与年龄有关的心脏健康下降的情况下，活化磺酰化将是有益的。更好地了解Ufl1与ufmylation靶标相互作用以及Ufsp1（潜在的Ufl1辅助因子；图）如何促进Ufl1的E3活性，可能会导致有关如何寻找或设计活化化合物的新思路。Uba5和Ufc1的激活剂也可能导致ufmylation的整体增加。相反，对Ufm1蛋白酶抑制剂的研究可能会鉴定出一种能够富集氨甲酰化蛋白质的化合物。最近的一份报告显示，细胞中Ufsp2的丢失会导致磺酰化靶标的急剧增加，[ ^16]提示抑制剂可能产生预期的效果。由于心力衰竭是发病率和死亡率的主要原因，尤其是在中年和老年人中，因此这项重要的研究表明，富马酰化是探索在心肌病和其他可能基于ER应激的疾病中获得治疗益处的有前途的途径。

Sutherland和Barrio博士由BFU2017-84653-P（欧盟MINECO / FEDER），Severo Ochoa卓越计划（SEV-2016-0644），UbiCODE计划（765445，欧盟）和PROTEOSTASIS（COST BM1307，欧盟）资助。巴斯克自治区政府工业，旅游和贸易部（Elkartek研究计划）和比斯开亚县创新技术部提供了额外的支持。

没有任何。

本文表达的观点不一定是编辑者或美国心脏协会的观点。

https://www.ahajournals.org/journal/circheartfailure